Rozprawy doktorskie na temat „Toll-like receptors”

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A endotoxemia é um importante distúrbio sistêmico que se origina da resposta do hospedeiro a um componente das bactérias Gram-negativas, o lipopolissacarídeo (LPS) ou endotoxina, que é liberado após bacteriólise ou rápida multiplicação. A ativação do sistema imune inato pelo LPS é um fator chave para o disparo da resposta inflamatória pelo hospedeiro e que acarreta a produção de mediadores inflamatórios, responsáveis pelos eventos patológicos da endotoxemia. A interação dos receptores Toll-like (TLRs) com antígenos específicos deflagram a resposta inflamatória, sendo que o receptor Toll-like-4 (TLR-4) é ativado pela ação das endotoxinas, enquanto o receptor Toll-like-2 (TLR-2) interage com uma variedade de componentes microbianos. Uma exposição prévia a baixas concentrações de LPS pode tornar os cavalos “tolerantes” a um desafio letal subsequente, acarretando uma diminuição na produção de citocinas inflamatórias por um período transitório. Pouco se sabe a respeito do mecanismo celular deste fenômeno em equinos, supondo-se o envolvimento dos receptores Toll-like semelhante ao encontrado em outras espécies. Com este estudo investigaram-se os mecanismos celulares da tolerância à endotoxina em um modelo ex vivo com sangue total. Foi demonstrado redução na síntese de citocinas pró-inflamatórias (TNF-α, IL-1 e IL-6), aumento da expressão gênica da citocina anti-inflamatória IL-10, e ausência de expressão do TGF-β, após o desafio secundário com LPS. A maior expressão dos receptores TLR-2 e -4 após o segundo estímulo de LPS demonstrou que a tolerância à endotoxina não acarreta diminuição da expressão de ambos os receptores em equinos.
Endotoxemia is an important systemic disease originated from host response to a component of Gram-negative bacteria, lipopolysaccharide (LPS) or endotoxin, which is released after bacteria death or quick replication. The innate immune recognition of LPS has a key role triggering host inflammatory answer and is due to inflammatory mediator’s synthesis, which are responsible for pathologic events of endotoxemia. Signs initiated by interaction of Toll-like receptors (TLRs) with specific products induce the inflammatory response. Toll-like receptor-4 (TLR- 4) is activated by endotoxin action while Toll-like receptor-2 (TLR-2) interacts with a range of microbial compounds. Some studies demonstrate that both can act like LPS receptors, although by independent pathways. It was demonstrated that a previous exposition to low concentrations of LPS can render horses “tolerant” to a lethal subsequent challenge with endotoxin, leading to a diminished release of inflammatory cytokines during a transient period. However, little is known about the cellular mechanisms involved in this phenomenon in horses, suspecting that there is involvement of cell surface receptors, similarly to other species. This study investigated cellular mechanisms of endotoxin tolerance in a whole blood ex vivo model, demonstrating a reduction on pro-inflammatory cytokines synthesis (TNF- α, IL-1 and IL-6), increased gene expression of anti-inflammatory cytokine IL-10 and no alteration in TGF-β expression, after a secondary stimulus with LPS. The Toll-like receptors-2 and -4 increased expression after a second stimulus with LPS showed that endotoxin tolerance does not lead to a decreased expression of both receptors in horses.

Naltrexone is an opioid antagonist usually used in the treatment of patients addicted to drugs or alcohol. However, since the 1980s naltrexone has been used at lower doses to treat patients with cancer and autoimmune diseases such as multiple sclerosis and Crohn’s disease. The mechanism of action of naltrexone in treating these diseases is unknown, however, evidence suggests that the drug has immune modulating effects. Toll like receptors (TLR) are type I membrane receptors that are crucial in the innate immune response. TLR recognise exogenous and endogenous ligands to induce the production of proinflammatory cytokines, chemokines and activation markers. Recent studies have suggested that naltrexone antagonises TLR4 and TLR9; providing an insight into the immunomodulatory ability of naltrexone. This thesis examines the effect naltrexone has on TLR expressed within the peripheral blood mononuclear cell (PBMC) population with primary focus on TLR 4 and TLR9. In this study, it was observed that naltrexone inhibits IL-6 produced by immune cell subsets following stimulation of TLR2, TLR7 and TLR9, but contrary to previous studies no inhibition of TLR4 was observed. Furthermore, it was determined that apoptosis is not induced under any condition. Studies examining isolated immune cell subsets demonstrated that naltrexone can modulate IL-6 production following stimulation of TLR7/8 on monocytes and TLR9 on B cells. However, naltrexone had no effect on B cell differentiation following stimulation with TLR9 ligand. This study is the first to examine the effect naltrexone has on human immune cells and the findings presented suggest naltrexone has the potential to modulate the production of cytokine in response to TLR activation. Therefore, this study provides preliminary evidence to support the hypothesis that naltrexone modulates TLR activity however, further research is required to justify the use of the drug as an immune modulator in patients with autoimmune diseases and cancer.

Patients with inflammatory or infectious conditions such as periodontitis, peri-implantitis, osteomyelitis, rheumatoid arthritis, septic arthritis and loosened joint prosthesis display varying severity of destruction in the adjacent bone tissue. Bone loss in inflammatory diseases is considered a consequence of cytokine induced RANKL and subsequent enhanced osteoclast formation. Hence, osteotropic cytokines and their receptors have been suggested to be important for the pathogenesis of inflammation-induced osteolysis. It is, here, suggested that bacterial components, so called “pathogen associated molecular patterns=PAMPs”, may also be involved. Varieties of cells express receptors for PAMPs, including Toll-like receptors (TLRs) which are the first line of defence in the innate immune system. LPS (lipopolysaccharide), fimbria and lipoproteins from pathogenic bacteria such as P. gingivalis, S. aureus are ligands for TLR2 and flagellin from pathogenic flagellated bacteria like S. typhimurium is a ligand for TLR5.   Since the susceptibility to, or the severity of inflammation-associated bone diseases are likely related to differences in the tissue response, and the mechanisms by which PAMPs interact with bone cells are not fully understood, we aimed to elucidate the importance of different TLRs for inflammation induced bone loss by conducting in vitro and in vivo investigations. Activation of TLR2 and TLR5 in organ cultured mouse parietal bones increased bone resorption in a time- and concentration-dependent manner by a process inhibited by OPG and bisphosphonate, showing the crucial role of RANKL-induced osteoclast formation. In addition, the number of osteoclasts, expression of osteoclastic genes and osteoclastogenic transcription factors were increased. In the bones and in osteoblasts isolated from the bones, TLR2 agonists increased the expression of RANKL without affecting OPG, while TLR5 activation resulted in enhanced RANKL and decreased OPG. Activation of both TLR2 and TLR5 stimulated the expression in both bones and osteoblasts of prostaglandins and pro-inflammatory cytokines, known to stimulate RANKL. By blocking the cytokines and prostaglandin, we showed that TLR2 and TLR5 induced bone resorption and RANKL expression are independent of these molecules. Activation of TLR2, but not TLR5, in mouse bone marrow macrophage cultures inhibited RANKL-induced osteoclast formation, an effect not observed in committed pre-osteoclasts. Local administration in vivo of TLR2 and TLR5 agonists on the top of mouse skull bones enhanced local and systemic osteoclast formation and bone resorption. Using knockout mice, we showed that the effects by LPS from P. gingivalis (used as TLR2 agonist) and flagellins (used as TLR5 agonists) are explicit for TLR2 and TLR5 ex vivo and in vivo, respectively. These data show that stimulation of TLR2 and TLR5 results in bone resorption in vitro and in vivo mediated by increased RANKL in osteoblasts and thus may be one mechanism for developing inflammatory bone loss. Interestingly, histological analyses of skull bones of mice treated locally with TLR2 and TLR5 agonists revealed that the bones not only reacted with locally increased osteoclastogenesis (osteoclast formation), but also with locally increased new bone formation. This was observed on both periosteal and endosteal sides of the bones, as well as in the bone marrow compartment. The formation of new bone was seen close to osteoclasts in some parts, but also in other areas, distant from these cells. The response was associated with active, cuboidal osteoblasts, extensive cell proliferation and increased expression of genes coding for bone matrix proteins and osteoblastic transcription factors. In conclusion, activation of TLR2 and TLR5 in osteoblasts results in bone loss associated with enhanced osteoclast formation and bone resorption, as well as with increased osteoblast differentiation and new bone formation, indicating that inflammation causes bone modeling. The data provide an explanation why LPS from P. gingivalis and flagellin from flagella-expressing bacteria can stimulate bone loss. Since TLR2 and TLR5 can be activated not only by bacterial components, but also by endogenous ligands produced in inflammatory processes, the data also contribute to the understanding of inflammation induced bone loss in autoimmune diseases. Hopefully, these findings will contribute to the development of treatment strategies for inflammation induced bone loss.

Las patologías funcionales (síndrome del intestino irritable, IBS) e inflamatorias gastrointestinales (enfermedad inflamatoria intestinal, IBD) se caracterizan por alteraciones de la función barrera epitelial, con un aumento de la permeabilidad, y cambios en la microbiota intestinal. Los receptores de tipo Toll (TLRs) participan en el reconocimiento bacteriano en el intestino y en el control neuroinmune local, estando, por tanto, implicados en la regulación de la función barrera del epitelio intestinal. El objetivo de este trabajo ha sido caracterizar la implicación de los receptores TLR5 y TLR7 en la regulación de la función barrera epitelial del colon. Para ello se ha caracterizado la función barrera epitelial, tanto en condiciones in vitro (electrofisiología y permeabilidad a macromoléculas en un sistema de cámaras de Ussing), como in vivo (permeabilidad a macromoléculas), tras la sobre-estimulación local de los receptores TLR5 y TLR7 con agonistas selectivos, flagelina e imiquimod, respectivamente, en rata y ratón. Los efectos en la función barrera se han caracterizado en condiciones normales, en estados de permeabilización del epitelio con DMSO, y en condiciones de inflamación (colitis inducida por dextrano sulfato de sodio -DSS-). Con la finalidad de definir el mecanismo de acción, se ha valorado la dinámica de las uniones estrechas epiteliales (expresión génica de proteínas -RT-qPCR- y distribución celular -inmunohistoquímica-) y la activación inmune local (expresión de citoquinas pro-inflamatorias). Los resultados obtenidos muestran que la sobre-estimulación del TLR7 del colon in vivo mejora la función barrera epitelial en la rata en condiciones fisiológicas, observando una reducción dosis-dependiente de la permeabilidad epitelial a macromoléculas evaluada en las cámaras de Ussing. No obstante, en condiciones de permeabilización del epitelio con DMSO, la sobre-estimulación del TLR7 causa un empeoramiento de la función barrera valorada in vivo. En ratones, la sobre-estimulación del TLR7 cólico in vitro no tiene efecto. Sin embargo, en un modelo de colitis inducida por DSS, reduce el aumento de la permeabilidad epitelial causado por la inflamación. Por tanto, parecen existir diferencias especie-específicas en los efectos de la sobre-estimulación del TLR7 cólico, pudiéndose observar tanto acciones promotoras como lesivas de la función barrera epitelial. La sobre-estimulación del TLR5 cólico agrava la disfunción de la barrera asociada a la inflamación (colitis inducida por DSS) en el ratón, incrementando la permeabilidad a macromoléculas. Sin embargo, la adición del agonista del TLR5, flagelina, en las cámaras de Ussing no afecta a la función barrera epitelial, ni en condiciones fisiológicas, ni durante la inflamación. En ningún caso, estos efectos moduladores de la función barrera se asociaron a cambios en la expresión génica de las principales proteínas de las uniones estrechas (claudina-2, claudina-3, ocludina, tricelulina, molécula de adhesión de la unión de tipo 1 y Zonula Occludens 1) ni a su distribución celular (claudina-2, claudina-3 y ZO-1). De la misma forma, los factores moduladores de la barrera, quinasa de la cadena ligera de la miosina y pro-glucagón (precursor del péptido similar al glucagón de tipo 2), tampoco presentaron cambios en su expresión asociados a la sobre-estimulación del TLR5 o del TLR7. Finalmente, se observó un efecto inmunomodulador receptor-específico. La sobre-estimulación del TLR7 reveló efectos potencialmente protectores al reducir la expresión de la citoquina pro-inflamatoria IL12-p40. Por el contrario, la sobre-estimulación del TLR5 tendió a aumentar la expresión de marcadores pro-inflamatorios, sugiriendo, por tanto, efectos pro-lesivos. En conclusión, estos resultados muestran la importancia de las interacciones microbiota-hospedador mediadas por TLRs en el control de la función barrera epitelial intestinal. Tanto el TLR7 como el TLR5 cólicos pueden considerarse potenciales dianas terapéuticas para el control de la función barrera y las respuestas inmunes locales en desórdenes funcionales e inflamatorios gastrointestinales como el IBD y el IBS.
Functional (irritable bowel syndrome, IBS) and inflammatory (inflammatory bowel disease, IBD) gastrointestinal disorders are characterized by an altered epithelial barrier function, with an increased permeability, and changes in the intestinal microbiota. Toll-Like Receptors (TLRs) participates in bacterial recognition within the intestine and in local neuro-immune control, thus participating in the regulation of intestinal epithelial barrier function. The objective of this work has been to characterize the implication of TLR5 and TLR7 in the regulation of colonic epithelial barrier function. For this, colonic epithelial barrier function has been studied in vitro (electrophysiology and permeability to macromolecules in a Ussing chamber system), as well as in in vivo conditions (permeability to macromolecules), after the local over-stimulation of TLR5 and TLR7 with selective agonists, flagellin and imiquimod, respectively, in rats and mice. The effects on barrier function have been studied in normal conditions, under states epithelial permeabilization with DMSO, and in conditions of inflammation -dextran sulfate sodium (DSS)-induced colitis-. In order to characterize the mechanisms of action, dynamics of tight junction (gene expression -RT-qPCR- and cellular distribution -immunohistochemistry- of tight junction proteins) and the presence of a local immune activation (gene expression of pro-inflammatory cytokines) were assessed. The results obtained indicate that the in vivo over-stimulation of colonic TLR7 improves epithelial barrier function in rats in physiological conditions, with a dose-dependent reduction in epithelial permeability to macromolecules, as assessed in Ussing chambers. However, under conditions of epithelial permeabilization with DMSO, the over-stimulation of TLR7 deteriorates barrier function, as assessed in vivo. In mice, the in vitro over-stimulation of colonic TLR7 was without effects. However, in a model of DSS-induced colitis, imiquimod reduces inflammation-induced increased epithelial permeability. Therefore, specie-specific differences seemed to exist for the barrier effects associated to the over-stimulation of colonic TLR7, leading to either protective or damaging actions on epithelial barrier function, depending upon the experimental conditions. The over-stimulation of colonic TLR5 aggravates the barrier dysfunction associated to inflammation (DSS-induced colitis) in mice, increasing the permeability to macromolecules. However, the direct addition of flagellin to the Ussing chambers did not affect epithelial barrier function, neither in physiologic conditions nor during inflammation. Regardless the conditions considered, TLR5/7-mediated modulatory actions on barrier function were not associated to changes in gene expression of the main tight junction-related proteins (claudin-2, claudin-3, occludin, tricellulin, junctional adhesion molecule 1 and Zonula Occludens 1). Moreover, no changes in the cellular distribution of tight junction proteins (claudin-2, claudin-3 y ZO-1) was observed. Likewise, TLR5/7 over-stimulation was not associated to changes in the expression of the barrier-modulating factors myosin light chain kinase and proglucagon (precursor of glucagon-like peptide 2). Finally, TLR-specific immunomodulatory effects were also observed. Over-stimulation of TLR7 revealed potential protective effects, reducing the expression of the pro-inflammatory cytokine IL12-p40. In contrast, over-stimulation of TLR5 tended to increase the expression of pro-inflammatory markers, thus suggesting pro-damaging effects. In conclusion, these results provide evidence of the importance of TLRs-dependent host-microbial interactions in the control of intestinal epithelial barrier function. Colonic TLR5 and TLR7 should be considered potential therapeutic targets for the control of barrier function and local immune responses in functional and gastrointestinal disorders, such as IBD and IBS.

Chemokines triggered by Toll-like receptors (TLRs) are small chemoattractant proteins, which mainly regulate leukocyte trafficking in inflammatory reactions via interaction with G protein-coupled receptors. Forty-two chemokines and 19 cognate receptors have been found in the human genome. Prior to this study, only 11 chicken chemokines and 7 receptors had been reported. The objectives of this study were to identify systematically chicken chemokines and their cognate receptor genes in the chicken genome and to annotate these genes and ligand-receptor binding by a comparative genomics approach. Twenty-three chemokine and 14 chemokine receptor genes were identified in the chicken genome. The number of coding exons in these genes and the syntenies are highly conserved between human, mouse, and chicken although the amino acid sequence homologies are generally low between mammalian and chicken chemokines. Chicken genes were named with the systematic nomenclature used in humans and mice based on phylogeny, synteny, and sequence homology. The independent nomenclature of chicken chemokines and chemokine receptors suggests that the chicken may have ligand-receptor pairings similar to mammals. The TLR family represents evolutionarily conserved components of the patternrecognizing receptors (PRRs) of the innate immune system that recognize specific pathogen-associated molecular patterns (PAMPs) through their ectodomains (ECDs). TLR's ECDs contain 19 to 25 tandem copies of leucine-rich repeat (LRR) motifs. TLRs play important roles in the activation of pro-inflammatory cytokines, chemokines and modulation of antigen-specific adaptive immune responses. To date, nine TLRs have been reported in chicken, along with a non-functional TLR8. Two non-mammalian TLRs, TLR21 and TLR22, have been identified in pufferfish and zebrafish. The objectives of this study were to determine if there is the existence of chicken genes homologous to fish-specific TLRs, and if possible ligands of these receptors exist. After searching the chicken genome sequence and EST database, a novel chicken TLR homologous to fish TLR21 was identified. Phylogenetic analysis indicated that the identified chicken TLR is the orthologue of TLR21 in fish. Bioinformatic analysis of potential PAMP binding sites within LRR insertions showed that CpG DNA is the putative ligand of this receptor.

Les récepteurs Toll like sont connus pour protéger de l'invasion des pathogènes et pour renforcer les réponses immunitaires. Notre objectif était d'étudier si le signal des TLRs est capable de réguler la lymphopoïèse B chez la souris. Nos résultats ont montré que les ligands de TLR2, TLR7 et TLR9 inhibent l'expansion in vitro des cellules pro-B. Nous avons étudié plus spécifiquement le TLR9, récepteur à l'ADN non méthylé contenant des motifs CpG. Le CpG inhibe l'expansion des cellules pro-B, mais n'a aucun effet sur les cellules pré-B. Le CpG inhibe l'expansion des cellules pro-B non pas par des facteurs solubles tels que les IFN de Type 1 mais par un effet direct. L'inhibition de l'expansion des cellules pro-B résulte d'une part de l'augmentation de l'apoptose et d'autre part de la réduction du nombre de cellules en cycle. L'apoptose des cellules pro-B induite par le CpG n'est pas une conséquence de l'inhibition du signal IL-7 ou d'un déséquilibre entre les membres de la famille Bcl-2. La transcription des gènes, connus pour inhiber la prolifération ou induire l'apoptose, n'était pas régulée par le CpG. Le signal TLR9 peut également réduire la lymphopoïèse in vivo, chez les souris SP6 Rag2-/- et les souris normales injectées avec le CpG. Les cellules B qui expriment le BCR nouvellement formé doivent franchir un point de contrôle. En effet, un BCR autoréactif est amené à changer sa spécificité par réarrangement des chaînes légères. Nous avons montré que dans le modèle transgénique SP6 qui présente un BCR anti-ADN, le signal TLR9 augmente le taux de réarrangement des chaînes légères et diminue au sein des IgMs sériques la fraction qui se lie à l'ADN
Toll like receptors are known as sentinels detecting pathogen invasion and as triggers of the Immune Response. We investigated whether TLR signaling regulates B cell lymphopoiesis and show that TLR2, TLR7 and TLR9 ligands inhibit the expansion of murine pro-B cells in vitro. We focused on TLR9 the receptor for unmethylated DNA containing CpG motifs, because such motifs, typical of microbial DNA, are also found in mammalian DNA. CpG inhibits the expansion of pro-B cells, by a mechanism dependent on MyD88. The inhibition is stage-specific because it only inhibits pro-B, but not pre-B, cell expansion. This inhibition is direct on the pro-B cells and not mediated by soluble factors, such as Type I IFN. CpG inhibits pro-B cell expansion, without interfering with the IL-7 signal, by inducing apoptosis and reducing the number of cells entering cell cycle. The apoptosis occurs through a mechanism independent of Bcl-2 family members and CpG does not promote transcription of genes known to repress proliferation or to induce apoptosis. TLR9 signaling can also reduce lymphopoiesis in vivo, as we observed in SP6 Rag2-/- mice and in wild type mice injected with CpG. Immature B cells express a newly formed BCR and must pass a checkpoint where the cells expressing an auto-reactive BCR are instructed to edit their receptors in order to lose self-reactivity. We sh6w that in the anti-DNA SP6 transgenic model, TLR9 signaling increases the editing of the light chain and decreases the fraction of serum IgM that binds to DNA. Therefore, TLR9 ligands, in addition to their role in the activation of effector cells, can also modulate the production and the specificity of B cells

Background: In the last two decades the discovery of the Toll-Like Receptor (TLR) family in humans revealed the importance of innate immunity in providing host defence against invading pathogens. Moreover, TLRs have a vital role in mediating the interactions between the reproductive and immune systems. Similar to TLRs, (NOD)-like receptors (NLRs); retinoic acid-inducible gene-1, RIG-1-like receptors (RLRs) are also important pattern recognition receptors in the female reproductive system. Successful implantation requires effective reciprocal interactions between receptive endometrium and competent embryo. The endometrial innate immunity during implantation has been intensively investigated. However, little is known about the expression of innate immunity during the early stages of human embryo development. Objective: To investigate the expression of innate immunity molecules during the early stages of human embryo development and to determine the functions of TLRs in blastocysts. Material and methods: The expression of TLRs, a panel of downstream signalling molecules, NLRs, RLRs, inflammatory cytokines, chemokines and the hyaluronic acid system were investigated in the following developmental stages: oocyte, 4- cell, blastomeres, 8- cell, blastocyst, inner cell mass and trophectoderm using Affymetrix Human Genome U133 Plus 2.0 arrays. Microarray data were validated by Q-PCR. TLR function in human blastocysts was investigated by treating day five blastocysts with TLR3 or TLR5 specific ligands; Poly (I:C) and flagellin respectively, for 24 hours. The culture media was analysed for elevated cytokine and chemokine levels using cytometric bead array. Results: TLRs, NLRs, RLRs, TLR downstream signalling molecules, cytokines and chemokines involved during implantation event and the hyaluronic system were all found to be positively expressed in the early stages of human embryo development. The expression levels of the above molecules were generally moderate to low (CT 24-34) and varied across the embryonic developmental stages. Stimulation of TLR3 and TLR5 in day 5 blastocysts produced cytokines and chemokines. In addition, there were alterations in gene expression patterns in the Poly (I:C) and flagellin treated blastocysts in comparison to the untreated blastocysts. Conclusion: The varied expression levels of the investigated molecules involved in early embryonic developmental suggests a potential role for these molecules in early pregnancy loss and implantation failure. Specifically, the relationship between the level of TLR expression, function and embryo quality is worth investigating in the future as a potential marker for embryo competence.

Orientador: Licio Augusto Velloso
Tese (doutorado) - Universidade Estadual de Campinas, FAculdade de Ciencias Medicas
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Resumo: O consumo de dietas ricas em gordura é o mais importante fator ambiental que contribui para o aumento da prevalência de obesidade na sociedade moderna. Em modelos animais com obesidade, induzida por dieta, a ativação da resposta inflamatória no hipotálamo produz resistência molecular e funcional aos hormônios anorexigênicos insulina e leptina, o que resulta em defeitos no controle da ingestão alimentar e do gasto energético. Para explorar a hipótese de que ácidos graxos podem desencadear resposta inflamatória e aumentar a expressão de citocinas no hipotálamo por induzir a ativação de TLR2/4 e/ou estresse de RE, ratos foram canulados e submetidos ao tratamento, por via intracerebroventricular (icv), com ácido graxo saturado e em seguida o extrato de proteínas hipotalâmicas foi obtido para determinação da expressão de proteínas por imunoblot. A localização de marcadores inflamatórios foi obtida por imunohistoquímica. Observou-se que ácidos graxos saturados de cadeia longa ativam predominantemente TLR4, o que determina não só a indução da expressão local de citocinas, mas também promove estresse de RE. Ratos alimentados com dieta hiperlipídica rica em ácidos graxos monoinsaturados não desenvolveram resistência hipotalâmica à leptina, enquanto que a mutação com perda da função do TLR4 e a inibição imunofarmacológica do TLR4 protegeu os camundongos da obesidade induzida por dieta. O TLR4 age como um alvo molecular predominante para ácidos graxos saturados no hipotálamo, o que desencadea resposta inflamatória e resistência a sinais anorexigênicos. Assim, conclui-se que ácidos graxos saturados ativam as vias de TLR2/TLR4 e estresse de RE em hipotálamo sendo a ativação mais significativa para TLR4, o que, por sua vez, determina a indução de estresse de RE. O TLR4 é um importante mediador da disfunção hipotalâmica durante o desenvolvimento da obesidade, como esse é um fenômeno importante durante a instalação da obesidade, esse receptor é um alvo terapêutico atrativo para tal condição epidêmica
Abstract: In animal models of diet-induced obesity, the activation of an inflammatory response in the hypothalamus produces molecular and functional resistance to the anorexigenic hormones insulin and leptin. The primary events triggered by dietary fats that ultimately lead to hypothalamic cytokine expression and inflammatory signaling are unknown. Here, we test the hypothesis that dietary fats act through the activation of Toll-like receptors 2/4 and endoplasmic reticulum stress to induce cytokine expression in the hypothalamus of rodents. Rats were treated icv with different types of fatty acids and hypothalamic protein extracts were obtained for determination of inflammatory and endoplasmic reticulum stress protein expression by immunoblot. Localization of inflammatory markers were performed by immunohistochemistry. Long-chain saturated fatty acids activate predominantly Toll-like receptor 4 signaling, which determines not only the induction of local cytokine expression but also promotes endoplasmic reticulum stress. Rats fed on a monounsaturated fat-rich diet do not develop hypothalamic leptin resistance, while Toll-like receptor 4 loss-of-function mutation and immunopharmacological inhibition of Toll-like receptor 4 protects mice from diet induced obesity. Toll-like receptor 4 acts as a predominant molecular target for saturated fatty acids in the hypothalamus, triggering the intracellular signaling network that induces an inflammatory response and determines the resistance to anorexigenic signals
Doutorado
Ciencias Basicas
Doutor em Clínica Médica

Orientador: Leonilda Maria Barbosa dos Santos
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: No presente estudo demonstramos a ativação de mecanismos imunosupressores em células dendríticas plasmocitóides (pDCs) e linfócitos B pela ação do agonista de TLR-9, ODN-CpG no modelo de estudo da esclerose múltipla (EM) e na encefalomielite experimental autoimune (EAE). A EAE é o modelo experimental da esclerose múltipla. Nossos resultados demonstram que a administração in vivo de ODN-CpG reduz significativamente a gravidade de EAE. A redução da doença foi acompanhada pela diminuição da resposta proliferativa dos linfócitos encefalitogênicos e consequentemente a infiltração dessas células no sistema nervoso central. A diminuição da resposta proliferativa parece ser devido ao efeito imunoregulador das pDCs, uma vez que a depleção dessas células faz com que a resposta proliferativa retorne aos níveis normais. O tratamento com ODN-CpG induziu a expressão da enzima indoleamine 2,3 dioxigenase pelas pDCs. Essa enzima está relacionada à geração de células T reguladoras. De fato nossos resultados mostraram um aumento da porcentagem de células T CD4+CD25+ e da expressão das citocinas anti-inflamatórias IL-10 e TGF-b no grupo tratado. Adicionalmente, a transferência de pDCs ativadas isoladas é capaz de reduzir a gravidade da doença. Além das pDCs, os linfócitos B também expressam TLR-9 e podem ser ativados pelo tratamento com CpG, de fato, embora o número de células não difira do controle não tratado, a transferência de linfócitos B de animais tratados com CpG é capaz de diminuir a gravidade da doença. O efeito supressor dos linfócitos B pode ser atribuído à expressão de IL-10 nos animais tratados. Em paralelo, nós demonstramos um aumento na porcentagem de pDCs em líquido cefalorraquidiano de pacientes com EM durante a fase de surto da doença quando comparados com pacientes em remissão ou com outras doenças neurológicas não inflamatórias. Nossos resultados indicam que elas podem estar envolvidas tanto com a piora da doença, o que poderia ser explicado por uma infecção viral, ou pelo contrário, estando em maior número poderia com sua ação imunomoduladora preparar o organismo para a fase de remissão da doença. Entretanto, pacientes com EM apresentam deficiência na indução de células T naive a produzirem IL-10, mas não IFN-g, o que poderia ser explicado em parte pela deficiência da expressão de IDO obervada após ativação in vitro com CpG, quando comparadas com pDCs de indivíduos saudáveis. A deficiência da expressão de IDO pode comprometer o efeito imunomodulador das pDCs na esclerose múltipla
Abstract: In the present study we verified the activation of immunomodulatory mechanisms of plasmacytoid dendritic cells (pDCs) and B lymphocytes by the action of TLR9 agonist, CpG-ODN during multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). EAE is the experimental model of human MS. Our results provide evidence that in vivo administration of CpG-ODN significantly reduces the severity of EAE. The reduction in disease was followed by decreasing in the proliferative response of encephalitogenic lymphocytes and consequently infiltration of these cells in the central nervous system. The decrease in the proliferative response seem to be due to the immunomodulatory effect of pDCs, since depletion of these cells restored the proliferative response, returns to normal levels. Treatment with ODN-CpG induced expression of indoleamine 2,3 dioxygenase enzyme by pDCs. This enzyme is related to the enhancement of regulatory T. Indeed, our results have shown an increased of percentage of CD25+CD4+Foxp3+ cells and expression of anti-inflammatory cytokines IL-10 and TGF-b in the treated group. Moreover, the adoptive transfer of activated pDCs alone reduced the clinical course of EAE. In addition to the pDCs, B lymphocytes also express TLR9 and can be activated by treatment with CpG, in fact, although the number of cells does not differ from untreated controls, the transfer of B lymphocytes from animals treated with CpG was able to reduce the severity of the disease as well. The immunomodulatory effect of B lymphocytes may be due to expression of IL-10 in treated animals. In parallel, we were able to report an increase of pDCs percentage in cerebrospinal fluid of MS patients during relapse compared with patients in remission or other non-inflammatory neurologic diseases. This data indicate that it may be involved with the worsening of the disease, which could be explained by viral infection, or be involved in the initial immunomodulatory mechanisms responsible for the remission. However, MS patients presented deficiency to induce naive T cells to produce IL-10, but not IFN-g, which could partly be explained by the deficiency of IDO expression observed after CpG in vitro activation, when compared with pDCs from healthy individuals. The deficiency of IDO expression may compromise the immunomodulatory effect of pDCs in MS disease
Doutorado
Ciencias Basicas
Doutor em Clínica Médica

Thesis (Ph. D.)--University of Missouri-Columbia, 2005.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "December 2005" Includes bibliographical references.

La leishmaniosis canina (CanL) es una enfermedad causada por el protozoa Leishmania infantum. Las manifestaciones clínicas en el perro son muy variables, abarcan desde una infección subclínica a un cuadro severo, que puede ser fatal. Al ser una enfermedad compleja, es difícil de diagnosticar. La respuesta inmunitaria de cada individuo juega un papel muy importante, tanto para el diagnóstico como para su tratamiento. Los receptores tipo Toll (TLRs) son los principales iniciadores de la respuesta inmunitaria innata cuya función es frenar cualquier intrusión de agentes extraños al cuerpo. Sin embargo, poca información existe sobre el papel de estos receptores en la CanL. El objetivo general de esta tesis doctoral es investigar el papel que los TLRs tiene en la CanL, tanto en experimentos ex vivo como in vitro. En la presente tesis doctoral, se ha estudiado la expresión genética en sangre no estimulada de los TLR2 y TLR4, en el momento del diagnóstico y durante el tratamiento, demostrando un notable aumento de la transcripción del TLR2 en perros con leishmaniosis clinica. Cuando se compararon perros enfermos en diferentes estadíos clínicos, se observó una expresión más notoria del TLR2 en aquellos perros que padecían la enfermedad de forma más severa comparado con los perros con estadíos más leves o con dermatitis papular. En, estos últimos, se notó de forma característica que tenían una alta producción de la citoquina interferon-gamma (IFN-γ). En sangre estimulada con antígeno soluble de Leishmania (LSA), la expresión del TLR2 y TLR4 disminuye en los perros enfermos que son productores de IFN-γ. Estimulando la sangre con TLRs agonistas, se halló un aumento en la producción de las citoquinas IFN-γ, TNF-α y IL-6 particularmente después de la estimulación con los agonistas TLR4 y TLR7 en sangre de perros naturalmente infectados. En el estudio in vitro incluido en esta tesis doctoral, en el cual macrófagos caninos fueron infectados experimentalmente con L. infantum y tratados con diferentes fármacos convencionales y TLRs agonistas, solos o combinados entre ellos, se demostró que la tasa de infección y la intensidad de infección se redujeron después de la estimulación los agonistas de TLRs. Además, el agonista TLR4 parece tener un efecto sinérgico combinado con el alopurinol. Así pues, con estos resultados, se ha ampliado el conocimiento de los TLRs en la infección por L. infantum en el perro. Y se han obtenido resultados esperanzadores para seguir investigando en esta línea sobre cómo los TLRs agonistas podrían actuar como inmunomoduladores en el tratamiento de esta enfermedad o incluso como adyuvantes en vacunas.
La leishmaniosi canina (CanL) és una malaltia zoonòtica causada pel paràsit Leishmania infantum, de distribució mundial i altament endèmica a la conca mediterrània. Aquest paràsit es transmet mitjançant la picada de les femelles de flebòtoms, sent el gos l'hoste i reservori principal. Les manifestacions clíniques de la infecció per L. infantum en el gos són molt variables, des d'una infecció subclínica crònica fins a una malaltia molt severa, que pot ser fatal. Deguda a la seva complexa patogènesi, és important el paper que juguen tant la resposta immunitària innata i adaptativa en aquesta infecció. No obstant això, hi ha més informació al gos sobre la resposta immunitària adaptativa que sobre la innata. Els receptors tipus Toll (TLRs) són primordials en la maquinària del sistema immunitari innat que faciliten la ràpida detenció de diverses infeccions així com l'activació de la cascada inflamatòria. No obstant això, el paper d'aquests receptors en la infecció per L. infantum en gossos no és molt coneguda i la informació que hi ha sobre els mateixos és molt limitada. La hipòtesi d'aquesta tesi doctoral és que els TLRs tenen un paper important en la infecció per L. infantum pel fet que estimulen la cascada inflamatòria. L'objectiu general de la present tesi doctoral va ser el d'investigar l'expressió de TLRs en la CanL comparant amb paràmetres parasitològics, clínics, bioquímics i immunològics així com avaluar el possible ús dels agonistes de TLRs en el tractament d'aquesta malaltia. Els objectius específics s'han desenvolupat en 5 estudis que es descriuen a continuació. El primer objectiu específic es descriu en el capítol 3.1. i va consistir en avaluar l'expressió dels receptors tipus Toll 2 (TLR2) i 4 (TLR4) en sang no estimulada de gossos amb leishmaniosi clínica moderada en el moment del diagnòstic i durant un any de tractament així com correlacionar els paràmetres clínics, bioquímics i parasitològics. En el capítol 3.2., es descriu el segon experiment on es va avaluar la quantificació relativa de l'TLR2 i TLR4 en gossos infectats per L. infantum en diferents estadis de la malaltia segons la classificació de LeishVet en el moment del diagnòstic. L'estudi va consistir a determinar a més els anticossos i la producció d'interferó gamma (IFN-γ) específiques enfront del paràsit així com la parasitèmia en estadi clínic I (gossos amb dermatitis papular) i en estadis clínics II-III (gossos amb dermatitis exfoliativa o ulcerativa). L'objectiu específic del tercer estudi (capítol 3.3.) Va ser el de determinar la transcripció dels gens TLR2, TLR4 i el lligant 1 de mort programada (PD-L1) en sang estimulada amb antigen soluble de L. infantum (LSA) i el mitogen Concanavalina a (Con A) en gossos malalts capaços de produïr IFN-γ i els no capaços i en gossos sans. El propòsit de l'estudi ex vivo 3.4. va ser determinar la producció de les citocines IFN-γ, TNF-α i IL-6 en sang estimulada amb LSA o agonistes TLRs (TLRsa) com el TLR3a, TLR4a i TLR7a sols o amb combinació de LSA i cadascun dels TLRa en gossos sans infectats naturalment per Leishmània. Finalment es va realitzar un experiment in vitro (estudi 3.5) on es va avaluar la susceptibilitat del paràsit a diferents fàrmacs convencionals anti-Leishmània (al·lopurinol, miltefosine o meglumine antimoniat) tan en promastigots com en amastigots en una línia cel·lular de macròfags canins així com amb el tractament dels agonistes de TLR2, 3, 4 i 7 sols o combinats amb els fàrmacs convencionals. Els estudis descriptius d'aquesta tesi han confirmat que els TLRs semblen tenir un paper en la malaltia. Els gossos infectats amb Leishmània malalts presenten diferent expressió dels TLRs comparat amb els individus sans, depenent directament també del perfil immunològic de l'animal i de l'estadi clínic. Així doncs, en aquesta tesi doctoral, es va observar la sobreexpressió del TLR2 en sang no estimulada en el moment del diagnòstic en gossos amb malaltia moderada (estadi II-III) en comparar amb els gossos sans. No obstant això, l'expressió de TLR2 en sang no estimulada va ser igual per als gossos amb malaltia lleu (estadi I) i els gossos sans. A més, es va produir una disminució de l'expressió del TLR2 durant un any de tractament i la millora clínica en els gossos amb malaltia moderada. No obstant això, no es van observar canvis en l'expressió de TLR4 en el diagnòstic ni durant el tractament en cap dels gossos estudiats. És important assenyalar també que l'expressió de TLR2 es va correlacionar amb paràmetres clínics, parasitològics i immunològics associats a malaltia de moderada a severa. A més, els gossos amb estadi lleu I i amb dermatitis papular tenien un perfil immunològic predominant Th1, en canvi, els animals classificats en estadis més severs predominava un perfil Th2. Els resultats obtinguts en sang estimulada amb LSA van ser molt interessants i correlacionats amb les troballes obtingudes en sang no estimulada. Es va observar una reducció en l'expressió gènica dels gens TLR2 i TLR4 en sang estimulada amb LSA en els gossos malalts que eren productors de IFN-γ comparat amb els gossos sans i una alta expressió de PD-L1 a tots els grups estudiats tant per LSA com per amb Con A. La sang estimulada amb TLRsa en gossos sans va resultar tenir una alta producció de les citoquines TNF-α i IL-6, comparades amb el medi sol. A més, es va observar un efecte sinèrgic pro-inflamatori quan es va estimular amb TLR4a i TLR7a en combinació amb LSA. En els estudis in vitro es va demostrar la susceptibilitat del paràsit als fàrmacs convencionals sent el fàrmac més eficaç la miltefosina. A més, es va demostrar que els TLRs agonistes sols redueixen la infecció i es va observar també sinergia en la reducció de la infecció amb al·lopurinol i els agonistes per al TLR4. No obstant això, no es va detectar la producció de TNF-α ni de NO en els sobrenedants recollits a les 72 hores. No obstant això, sí que es va detectar la transcripció dels TLR2, 4 i 7 en totes les condicions estudiades. En general, es va demostrar una disminució de la transcripció de TLR2 o sense canvis en l'expressió de TLR4 i TLR7 amb la infecció . No obstant això, l'expressió de TLRs després del tractament amb fàrmacs anti-Leishmania convencionals sols o els agonistes de TLRs sols o combinacions dels dos va ser més variable. En conclusió, aquesta tesi doctoral ha demostrat que l'expressió de TLR2 en sang no estimulada és un marcador de malaltia de moderada a severa. No obstant això, el TLR4 no sembla ser un bon marcador per CanL en sang no estimulada. A més, la reducció de l'expressió dels TLR2 i 4 en sang estimulada amb LSA es va associar a gossos malalts productors d’IFN-γ els quals tenen un perfil més protector que gossos no productors d’IFN-γ. L'experiment in vitro ens va revelar que els fàrmacs combinats amb els TLRsa o fins i tot els TLRsa sols poden reduir la infecció. Per aquest motiu, els resultats trobats en aquesta tesi suggereixen que els TLRs podrien utilitzar-se com immunoteràpia, sols o combinats amb fàrmacs convencionals.
Canine leishmaniosis (CanL) is a zoonotic disease caused by the protozoan Leishmania infantum, of worldwide distribution and highly endemic in the Mediterranean basin. This parasite is transmitted by the bite of sandfly females. The dog is the main host and reservoir. The clinical manifestations of L. infantum infection in the dog are very variable and range from a chronic subclinical infection to a very severe disease, which can be fatal. Due to its wide pathogenesis, the innate and adaptive immune responses play a role in this canine infection. However, there is much more information on the adaptive immune response than on the innate one. Toll-like receptors (TLR) are essential in the machinery of the immune system that facilitates the early arrest of several infections as well as the activation of the inflammatory cascade. However, the role of these receptors in L. infantum infection in dogs is not well known and the information is very limited. The hypothesis of this doctoral thesis is that TLRs have an important role in L. infantum infection in dogs because they stimulate the inflammatory cascade. The general objective of this doctoral thesis was to investigate the expression of TLRs in the CanL and compare with parasitological, clinical, biochemical and immunological parameters as well as to evaluate the possible use of TLR agonists in the treatment of this disease. The specific objectives have been developed in five studies and are described below. The first specific objective is described in chapter 3.1. and consisted in evaluating the expression of Toll-like receptors 2 (TLR2) and 4 (TLR4) in unstimulated blood of dogs with moderate clinical leishmaniosis at the time of diagnosis and during one year of treatment as well as correlating clinical, biochemical and parasitological parameters. In chapter 3.2., we described the second experiment where the relative quantification of TLR2 and TLR4 was evaluated in dogs infected by L. infantum in different stages of the disease according to the LeishVet classification at the time of diagnosis. This study also investigated the antibodies and the production of interferon gamma (IFN-γ) specific to the parasite as well as the parasitemia in clinical stage I (dogs with papular dermatitis) and in clinical stages II-III (dogs with exfoliative dermatitis or ulcerative) The specific objective of the third study (Chapter 3.3.) was to determine the transcription of the TLR2, TLR4 and programmed death ligand 1 (PD-L1) genes in blood stimulated with L. infantum soluble antigen (LSA) and the mitogen Concanavalin A (Con A) in sick dogs IFN-γ producers and non-IFN-γ producers and healthy dogs. The purpose of the study described in chapter 3.4. was to determine the production of the cytokines TNF-α and IL-6 in blood stimulated with LSA or TLRs agonists (TLR3, TLR4 and TLR7) alone or combined of dogs naturally infected by Leishmania. Finally, an in vitro experiment (Chapter 3.5) was carried out where the parasites’ susceptibility to different conventional anti-Leishmania drugs (allopurinol, miltefosine or meglumine antimonate) was evaluated in promastigote and amastigote assays in a canine macrophage cell line as well as in the treatment of TLRs agonists 2, 3, 4 and 7 (TLRsa) alone or in combination with conventional drugs. Descriptive studies of this thesis have confirmed that TLRs seem to have a role in the disease. Sick dogs infected with Leishmania present different expression of TLRs compared to healthy individuals, also directly depending on the immunological profile of the animal as well as the clinical stage. Thus, in this doctoral thesis, overexpression of TLR2 was obtained in unstimulated blood at the time of diagnosis in dogs with moderate disease (stage II-III) when compared with healthy dogs. However, expression of TLR2 in unstimulated blood was the same for dogs with mild disease (stage I) and healthy dogs. In addition, there was a decrease in the expression of TLR2 during one year of treatment and clinical improvement in dogs with moderate disease. However, no changes were observed in TLR4 expression at diagnosis or during treatment in any of the dogs studied. It is also important to highlight that the expression of TLR2 was correlated with clinical, parasitological and immunological parameters associated with moderate to severe disease. In addition, dogs with mild stage I and papular dermatitis had a predominantly Th1 immunological profile whereas animals classified in more severe stages had a Th2 profile predominant. The results obtained in blood stimulated with LSA were very interesting and correlated with the findings obtained in unstimulated blood. A reduction in gene expression of the TLR2 and TLR4 genes in blood stimulated with LSA was observed in sick dogs that were IFN-γ producers compared to healthy dogs and a high expression of PD-L1 in all the groups studied for both LSA as for Con A. The blood stimulated with TLRs agonists (TLRsa) and LSA of sick dogs turned out to have a high production of the cytokines IFN-γ, TNF-α and IL-6, compared with the medium alone. The combinations that gave the most production of cytokines of the Th1 profile are the agonists TLR4 and TLR7 each combined with LSA. In vitro studies demonstrated the susceptibility of the parasite to conventional drugs, being miltefosine the most effective drug. In addition, it was shown that TLRsa agonists alone reduced infection, and a synergistic effect was also observed in the reduction of infection with allopurinol and agonists for TLR4. However, the production of TNF-α or NO was not detected in the supernatants collected after 72 hours. However, the transcription of TLR2, 4 and 7 was detected in all the conditions studied. In general, a decrease in the transcription of TLR2s was demonstrated or no changes in the expression of TLR4 and TLR7 with infection. However, the expression of TLRs after treatment with conventional anti-Leishmania drugs alone or TLR agonists alone or combination of both was more variable. In conclusion, this doctoral thesis has shown that the expression of TLR2 in blood is not stimulated in a marker of moderate to severe disease. However, TLR4 does not appear to be a good marker for CanL in unstimulated blood. In addition, the reduced expression of TLR2 and 4 in blood stimulated with LSA was associated with sick dogs responding to IFN-γ which have a more protective profile than dogs not responding to IFN-γ. The in vitro study revealed that drugs combined with TLRsa or even TLRsa alone can reduce infection. For this reason, the findings found in this thesis are that TLRs can be used as immunotherapy or as adjuvants in future vaccines.

Hepatitis C Virus (HCV) is the primary cause of liver transplantation due to its chronic nature in up to eighty percent of infected cases. Around 3 percent of the world’s population is infected with HCV. Treatment for HCV is a combined Ribavirin and interferon-α (IFN-α) therapy effective in only fifty to eighty percent of patients depending on HCV genotype. The growing health concern with this disease is the lack of a cure despite liver transplantation. HCV targets hepatocytes, liver cells, but is not cytolytic. HCV has been shown to induce end stage liver disease through sustained inflammation from the host’s immune system in the liver. One of the key dilemmas in HCV research and the search for fully effective treatments or vaccines is the lack of animal models. HCV infectivity and disease is limited to primates, most specifically to humans, which cannot be fully replicated in any other living being. The mechanisms for HCV evasion or activation of the immune system are complex, many and discoveries within this field are crucial to overcoming this destructive hepatic infection. Toll-like receptors (TLR) are cellular activators of the innate immune system that have been a target of HCV. Activated TLRs trigger both the inflammatory and anti-viral pathways to produce inflammatory cytokines and interferons. HCV proteins have been reported to activate a number of TLRs in a variety of cell types. In order to identify possible targets of HCV within the TLR family, we first characterized TLR presence and function in both human hepatic carcinoma cell lines and purified primary human hepatocytes. RNA from TLRs 1-10 was observed to varying degrees in both the hepatoma cell lines and the primary hepatocytes. We show the extracellular and/or intracellular presence of TLR2, TLR1, TLR3 and TLR7 proteins in hepatoma cell lines. TLR3 and TLR7 are located within the endosome and recognize viral RNA products. We recently reported that TLR2-mediated innate immune signaling pathways are activated by HCV core and NS3 proteins. TLR2 activation requires homo- or heterodimerization with either TLR1 or TLR6. We show NF-κB activation in hepatoma cells by TLR2/1, TLR2/6 ligand and HCV protein stimulation. In primary hepatocytes, HCV proteins induced both IL-8 and IL-6 production. We also show that primary hepatocytes initiate a Type 1 IFN response in addition to IL-8 and IL-6 production upon stimulation with a TLR7/8 ligand. Human hepatoma and primary hepatocytes are responsive to TLR2, TLR1, TLR6, TLR7/8 ligands and HCV proteins. Activation of these TLRs may contribute to the inflammatory mediated destruction caused by HCV or could be targets of HCV contributing to its immune evasion. We found previously that hepatoma cells and primary hepatocytes are responsive to TLR2 ligands and HCV proteins. We also reported that TLR2 is activated by HCV proteins. Here we aimed to determine whether TLR2 coreceptors participated in cellular activation by HCV core or NS3 proteins. By designing siRNAs targeted to TLR2, TLR1 and TLR6, we showed that knockdown of each of these receptors impairs pro- and anti-inflammatory cytokine activation by TLR-specific ligands as well as by HCV core and NS3 proteins in Human Embryonic Kidney cells (HEK/TLR2) and in primary human macrophages. We found that HCV core and NS3 proteins induced TNF-α and IL-10 production in human monocyte-derived macrophages, which was impaired by TLR2, TLR1 and TLR6 knockdown. Contrary to human data, results from TLR2, TLR1 or TLR6 knockout mice indicated that the absence of TLR2 and its coreceptor TLR6, but not TLR1, prevented the HCV core and NS3 protein-induced peritoneal macrophage activation. TLR2 may utilize both TLR1 and TLR6 coreceptors for HCV core- and NS3-mediated activation of macrophages and innate immunity in humans. These results imply that multiple pattern recognition receptors could participate in cellular activation by HCV proteins contributing to inflammatory disease. Two critical factors in chronic HCV infection are inflammatory disease and immune evasion. We have demonstrated that TLR2 and its co-receptors play a role in inflammatory-mediated induction via HCV NS3 and core administration. It has recently been shown that HCV targets the TLR3 pathway to aid in immune evasion. TLR3 is only one of four viral recognition receptors located within the endosome and it is plausible that HCV may target others. We hypothesized that HCV infection may interfere with the expression and function of TLR7, a sensor of single stranded RNA. Investigating any effect on TLR7 by HCV may reveal a new mechanism for HCV immune evasion. Low levels of both TLR7 mRNA and protein were measured in HCV replicating cells compared to control cells while reducing HCV infection with either IFNα or restrictive culture conditions restored the decreased TLR7 expression. Downstream of the TLR7 pathway, an increased baseline IRF7 nuclear translocation was observed in HCV replicating cells compared to controls. Stimulation with a TLR7 ligand, R837, resulted in significant IRF7 nuclear translocation in control cells. In contrast, HCV replicating cells showed impaired IRF7 activation. Use of RNA polymerase inhibitors on hepatoma cells, control and HCV replicating, revealed a shorter TLR7 half life in HCV replicating cells compared to control cells which was not seen in TLR5 mRNA. These data suggest that reduced TLR7 expression, due to RNA instability, directly correlates with HCV replication and results in impaired TLR7-induced IRF7-mediated cell activation. In conclusion, Hepatitis C Virus manipulates specific Toll-like receptors’ expression and their signaling pathways to induce cytokine production. HCV utilizes surface receptors TLR2 and its co-receptors which once activated could contribute to inflammatory disease by production of inflammatory cytokines and possibly immune evasion. HCV down-regulates TLR7, a viral recognition receptor, by decreasing mRNA stability which could facilitate evasion of host immune surveillance.

Recently, it has been demonstrated that invasive but nonpathogenic Salmonella typhimurium i.t. injected into melanoma B16-bearing mice led to complete regression of the tumor, although Salmonella was unable to directly kill B16 tumor cells in vitro. Since we found high levels of IFN-γ in infected tumor masses in vivo, we wondered whether this cytokine might be involved in tumor cell death. In this work, we have demonstrated that IFN-γ shows a cytotoxic effect only when administered in combination with Salmonella. In addition, this combined treatment is able to enhance TLR transcription, mainly TLR2 and TLR3, in B16 cells, suggesting a possible link between tumor cell death and TLRs, as already proposed in some studies. Our hypothesis is strengthened by the finding that, unlike IFN-γ, IFN-α does not induce neither tumor cell death nor TLR transcription. B16 tumor cell death induced by either TLR2 ligand Pam3SCK4 or TLR3 ligand poly I:C treatment in combination with IFN-γ indicates that both TLR2-TLR1 heterodimer and TLR3 are involved in tumor cell death, although they likely activate independent mechanisms. On the contrary, neither TLR2-TLR6 nor TLR4 seems to be able to efficiently kill tumor cells neither alone nor in combination with IFN-γ. Notably, several evidences suggest that Salmonella in combination with IFN-γ activates TLR2 rather than TLR3. However, since we cannot definitely rule out the existence of a TLR3 ligand in Salmonella, further experiments using anti-TLR2 and anti-TLR3 blocking antibodies will be necessary. Furthermore, in this study we demonstrate that B16 tumor cells undergo a first necrotic event in the first 24 hours of combined stimulations inducing IL-6 and KC release, probably through NF-kB activation. Moreover, caspase activation analysis has demonstrated that B16 cells undergo apoptosis as well, but a later time. In this regard, we detected mainly caspase-7 cleavage after 48 hours of combined treatments. However, incubation with the necrosis inhibitor necrostatin-1 (nec-1) strongly reduces caspase-7 activation, suggesting that a necrotic event might be responsible for the subsequent apoptosis, that may be independent of TLR engagement. B16 dying cells release HMGB1 as well, a marker of necrotic cell death, that is able to enhance tumor immunogenicity. However, since Salmonella alone triggers HMGB1 release without inducing cell death, HMGB1 may be actively secreted in response to Salmonella, but may also be passively released by necrotic dying cells. Because of the important role of HMGB1 in tumor immunogenicity, future studies will be performed with the aim of understanding how B16 cells release HMGB1. Our results suggest that this may be achieved through a TLR4 MyD88-independent pathway. Interestingly, we demonstrate for the first time that poly I:C induces TLR3 translocation to B16 cell surface in combination with IFN-γ. In this way, poly I:C can improve TLR3-mediated response thus probably inducing a stronger caspase-7 activation with respect to Salmonella and TLR2 ligand. Finally, different experiments have highlighted the importance of IFN-γ in this tumor cell death mechanism. In particular, IFN-γ seems to sensitize tumor cells before receiving a subsequent stimulation, up-regulating TLRs involved in tumor cell death (e.g. TLR3) or through other unknown mechanisms. These results set the basis to improve the immunotherapy protocol developed in Maria Rescigno’s laboratory, combining IFN-γ i.t. injection with Salmonella i.t. injection or replacing Salmonella with Pam3CSK4, in order to obtain a more prompt therapeutic effect avoiding possible side effects of Salmonella. In addition, these results provide a rationale for introducing poly I:C treatment into electrochemotherapy approach, a new method applied to cutaneous cancers treatment, melanoma included. Since it is based on the permeabilization of the cell membrane by means of short and intense electric pulses, it would allow the entry of poly I:C into the cytosol of both tumor and immune cells. Therefore, TLR3 ligands may be involved in both the killing of tumor cells and in activating the immune system. Importantly, this new system may overcome the use of a systemic treatment with IFN-γ and could be used to treat melanoma avoiding possible side effects.

Thesis (Ph. D.)--University of Missouri-Columbia, 2008.
"May 2008" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

Antigen recognition by B cells results in their activation followed by specific antibody production. These events are initiated by antigen binding to their surface B cell receptors (BCR) which triggers both signalling and internalization of the receptor bound antigen to the endosome. However B cells also express features of the innate immune system such as Toll like receptors (TLRs), that can be located either on the surface of the cell or intracellularly where they recognize bacterial and viral nucleic acids. Engagement of these receptors within B cells is associated with enhancement of humoral responses. The aim of my PhD project was to investigate how endosomal TLR ligands in a particulate form could gain access to their intracellular receptors in the B cell and which impact the subsequent TLR engagement had on B cell fate. To achieve this, I directly linked both antigen and TLR9 ligand to particulates. Immunisation of mice with those particulates resulted in enhanced specific antibody titers compared to stimulation with particulate antigen alone. To dissect the underlying mechanism, I employed transgenic B cells bearing BCR specificity for the same antigen and stimulated them with particulate antigen-TLR9 ligand conjugates. Particulate TLR9 ligand could not gain access to its receptor within B cells via unspecific macropinocytosis and instead depended on BCRmediated internalization. Subsequent engagement of intracellular TLR9 by its ligand present in the conjugates resulted in B cell activation and proliferation, followed by differentiation into plasma cells and antigen specific antibody secretion. The uptake of the antigen-TLR9 ligand particulates both in vitro and in vivo depended on the affinity of the antigen once a defined threshold required for internalization was surpassed. The extent of plasma cell differentiation however could be modulated by the amount of TLR9 ligand present on the particulates. Thus I observed that direct linking of antigen and TLR ligand resulted in PC differentiation through antigen specific BCR mediated internalization and subsequent TLR engagement. This reveals a mechanism that may operate during the initiation of a primary immune response.

Abstract Incidence of esophageal adenocarcinoma is rising rapidly in Western countries. The main risk factor for esophageal adenocarcinoma is Barrett’s esophagus. Barrett’s esophagus results from long-term gastroesophageal reflux disease. The gastrointestinal tract is colonized by bacteria, fungi and viruses forming the alimentary tract microbiome. Microbiome transformation is involved in pathogenesis of alimentary tract cancer and also in the development of Barrett’s metaplasia. Toll-like receptors (TLR) are molecules of the innate immune system and they are involved in bacterial and viral recognition and regulation of immune functions in the host and cancer cells. This thesis examined the effect of alimentary tract microbiome and cancer- on the function of TLRs in normal gastrointestinal epithelial cells. An additional focus of the thesis was also to assess the carcinogenetic effect of TLRs 1–9 in Barrett’s esophagus metaplasia – dysplasia – carcinoma sequence. Study material consisted of: a patient cohort, organ donors, conventional and germ-free mice. TLRs are expressed also in a “microbe-free” gut. There were significant differences in all TLRs between small- and large intestine of conventional mice and in humans. In germ-free mice that difference was not observed. Normal tissue sampled adjacent to the tumors of cancer patients can be used as controls in immunohistochemical TLR studies in gastrointestinal cancer Clinical data indicate that TLRs linearly increase toward dysplasia in Barrett’s esophagus. High cytoplasmic and nuclear TLR4 expression and TLR1 and 8 nuclear immunoreactivity in esophageal adenocarcinoma are associated with metastatic disease and poor prognosis. Based on our results, bacteria seem to downregulate TLR expression of the intestine. TLRs 1–9 apparently have a role in malignant progression of Barrett’s dysplasia. TLR1, TLR4 and TLR8 may represent a novel therapeutic target in esophageal adenocarcinoma
Tiivistelmä Ruokatorven adenokarsinooma on länsimaissa nopeasti yleistyvä syöpätyyppi. Tämän syöpätyypin tärkein riskitekijä on Barrettin ruokatorvi, joka kehittyy pitkään jatkuneen gastroesofageaalisen refluksitaudin pohjalta. Ruuansulatuskanavassa on suuri määrä bakteereja, sieniä ja viruksia, jotka muodostavat yhdessä ruuansulatuskanavan mikrobiomin. Normaalin mikrobiomin muutokset ovat yhteydessä usean eri ruuansulatuskanavan syövän patogeneesiin ja myös Barrettin ruokatorven muodostumiseen. Tollin kaltaiset reseptorit ovat luontaisen immuniteetin molekyylejä, jotka osallistuvat bakteerien ja virusten tunnistukseen ja sääntelevät immuunivastetta sekä normaalitilanteessa että syövissä. Väitöskirjassa tutkitaan ruuansulatuskanavan mikrobiomin ja syövän vaikutuksia normaalien epiteelisolujen TLR:ien toimintaan. Lisäksi selvitetään TLR:ien karsinogeneettisiä vaikutuksia Barrettin ruokatorven metaplasia- dysplasia -adenokarsinoomasekvenssissä. Tutkimusmateriaalina käytetään potilaskohortista ja elinluovutuksista peräisin olevia potilasnäytteitä sekä normaalien ja bakteerittomien hiirien näytteitä. Tuloksemme osoittavat, että TLR:t ilmentyvät myös bakteerittomassa ruuansulatuskanavassa, ja TLR:en ilmentyminen oli merkittävästi voimakkaampaa ohutsuolessa kuin paksusuolessa normaaleilla hiirillä ja ihmisillä. Tätä eroa ei havaittu bakteerittomilla hiirillä. Ruuansulatuskanavan syöpien viereistä ja sen altistamaa tervettä kudosta voidaan käyttää terveenä kontrollina immunohistokemiallisissa TLR- tutkimuksissa. Kliinisessä aineistossa TLR:ien ilmentyminen kasvaa lineaarisesti kohti dysplasiaa Barrettin ruokatorvessa. TLR4:n korkea ilmentyminen solulimassa ja tumassa sekä TLR8:n ilmentyminen tumassa ovat yhteydessä metastaattiseen tautiin ja huonoon ennusteeseen. Tulosten perusteella bakteerit näyttävät heikentävän TLR:ien toimintaa suolistossa. Lisäksi kaikilla tutkituilla TLR:illä (1–9) näyttää olevan osuutta Barrettin dysplasian etenemisessä kohti syöpää. TLR1, TLR4 ja TLR8 ovat mahdollisia terapeuttisia kohteita ruokatorven adenokarsinoomassa

PURPOSE- Antimicrobial peptides (AMPs) are cationic host defence peptides with microbicidal properties providing a "chemical barrier" in response to pathogens and various pathological states. They show promise as potential therapeutic agents. Amniotic membrane (AM) transplantation is indicated in ocular surface surgery procedure. However, little is known of the AMP profile and variability within the AM. In addition, the effects of processing, storage, and preoperative preparation on AMPs in the transplant ready amniotic membrane (TRAM) are not clear. This can alter the therapeutic potential of AM used for such procedures. In the present study, we first profiled the expression of AMPs on the amniotic membrane in healthy women delivering by caesarean section. This was followed by protein analysis of one a potent AMP namely HBD-3 in fresh AM versus TRAM. Methods- Amniotic membrane samples of healthy women delivering by elective caesarian section were studied. Conventional PCR (semi-quantitative RT- PCR) of 21 AMPs was performed. Subsequently, quantitative real-time polymerase chain reactions (QRT-PCR), confirmed the relative gene expression of eight AMPs, by RT- PCR in these healthy patients. Further, the AMPs were confirmed to be primarily localized to the epithelium by immunohistochemistry. Western blotting in fresh AM with spongy layer, AM alone and TRAM and antimicrobial activity of TRAM was also performed. Results- RT- PCR showed expression of 8AMPs namely HBD-1, HBD-2, HBD- 3, LL37, Leap-1, Leap-2 DEFB109 and RNase7 in variable patterns. QRT-PCR confirmed expression of HBD-3 (mean 1.11; +/- 0.05, SEM 0.02), LEAP-2 (mean 1.20; +/- 0.02, SEM 0.01), and DEFB109 (mean 1.07; +/- 0.06, SEM 0.03), in all five samples with relatively little variation in the normalized expression of these three genes. HBD-1, HBD-2 and RNase7 were also expressed but in fewer (2 /5, 4/5, 3/5) samples respectively. LL37 and Leap-1 were detected in only one sample (AM 51) and confirmed by QRT- PCR in another 10 fresh samples. All 10 TLRs were noted to be expressed by RT- PCR. QRT-PCR confirmed expression of TLR-1 (mean 1.32; +/- 0.03, SEM 0.01) and TLR-3 (mean 1.21; +/- 0.07, SEM 0.03) in all five samples. While TLR-6 (mean 1.16; +/- 0.03, SEM 0.01) and TLR-7 (mean 1.31; +/- 0.09, SEM 0.05) were present in most (4/5) samples, TLR 2 (mean 1.29; +/- 0.02, SEM 0.06),TLR-4 (mean 1.42; +/- 0.05, SEM 0.02), TLR-5 (mean 1.22; +/- 0.1, SEM 0.01) and TLR-10 (mean 1.19; +/- 0.03, SEM 0.07) were noted to be expressed in only 3/5 samples. While TLR 8 (mean 1.19; +/- 0.07, SEM 0.1) was noted in two samples, TLR 9 was expressed in only one sample. Western blotting was unable to detect the presence of these small peptides. Immunohistochemistry confirmed the presence of these 8 peptides in fresh and TRAM AM epithelium but not in spongy layer. Both fresh and TRAM were not noted to have any significant antimicrobial properties. Conclusion- In this study, we profiled the AMP expression on the AM surface. We detected the expression of novel AMPs, Leap-2 and DEFB109 constitutively in all AM samples, along with confirming the constitutive expression of HBD-3. We also found a variable expression of RNase7, 6-defensins HBD-1, HBD-2, and AMPs LL-37 and LEAP-1 on the AM surface. We found that, while AMPs are localized in the epithelium they are absent in compact and spongy layer of the amniotic membrane. There are inter-donor variations but no significant intra-donor variations within regions. While AMP genes are expressed by the AM surface, they may not reach therapeutic potential as noted by the absence of the antimicrobial properties of TRAM by both MIC and MBC. The expression of these small but important peptides (potentially all other soluble proteins at the AM surface) may be altered by current processing techniques. Leap-2 and DEFB109 alongside HBD-3 may have a role in developing the innate immune response in the foetus.

Thesis (Ph.D. )--University of Tennessee Health Science Center, 2007.
Title from title page screen (viewed on May 16, 2008 ). Research advisor: Gerald I. Byrne, Ph.D. Document formatted into pages (xi, 114 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 65-107).

Adrenal gland insufficiency – the clinical manifestation of deficient production or action of adrenal steroids – is a life-threatening disorder. Among many factors which can predispose to primary adrenal failure, an autoimmune adrenalitis and infectious agents play a major role. The initial host defense against bacterial infections is executed primarily by the pattern recognition receptors, e.g. Toll-like receptors (TLRs), expressed in cells from the innate immune system. Upon activation, TLRs have been found to regulate various levels of innate and adaptive immunity as well as control tissue inflammation. TLRs are implicated in adrenal cell turnover and steroidogenesis during inflammation. Therefore, TLRs play a crucial role in the activation of adrenal inflammation mediating adrenal gland dysfunction during septicemia
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Chronic Lymphocytic Leukaemia (CLL) is the most common form of Leukaemia in the Western world and has a highly variable clinical course. Continuing advances in the range of therapeutic options available to clinicians require reliable prognostic indicators that can be used to group patients accurately according to their risk of disease progression, thereby allowing meaningful comparisons of treatments. The expression of Toll-Like Receptors (TLR) on cells involved with the disease process in CLL was studied to establish links between levels of expression and the disease process. The expression levels of 5 different TLR were measured on a variety of haemic cells and compared with the TLR expression levels seen on their normal counterparts. Flow cytometric analysis was used to establish the expression levels of TLR 1,2,3,4, and 9 on peripheral blood Monocytes, T Lymphocytes and B Lymphocytes from 129 patients. These results were compared with the TLR expression on corresponding cells from an equal number of age and sex matched controls. Further studies were performed which established the detrimental effect that storage of samples has on TLR expression, and also to compare TLR expression in patients who exhibited a positive Direct Antiglobulin Test (DAGT), with those that were negative for the DAGT. Results from the study show that both T and B lymphocytes from CLL patients showed statistically significantly different levels of TLR expression when compared with lymphocytes from age and sex matched controls. TLR expression levels on monocytes were similar in both patient and control groups. When comparing TLR expression between patients who were DAGT positive and those that were negative, a statistically significant difference was found in TLR9 expression on T lymphocytes. These findings have established that that there are statistically significant differences in TLR expression on lymphocytes when comparing CLL patients with age and sex matched controls. It also establishes the differences in TLR expression levels seen in DAGT positive and DAGT negative patients. From findings made during this study, it is hypothesised that there may be a link between differential TLR expression and the autoimmune disease frequently reported in CLL.

Adrenal gland insufficiency – the clinical manifestation of deficient production or action of adrenal steroids – is a life-threatening disorder. Among many factors which can predispose to primary adrenal failure, an autoimmune adrenalitis and infectious agents play a major role. The initial host defense against bacterial infections is executed primarily by the pattern recognition receptors, e.g. Toll-like receptors (TLRs), expressed in cells from the innate immune system. Upon activation, TLRs have been found to regulate various levels of innate and adaptive immunity as well as control tissue inflammation. TLRs are implicated in adrenal cell turnover and steroidogenesis during inflammation. Therefore, TLRs play a crucial role in the activation of adrenal inflammation mediating adrenal gland dysfunction during septicemia.
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Introdução: Um dos primeiros sistemas de defesa contra os microrganismos é a via dos receptores Toll-like (TLRs). A ativação destes receptores leva à síntese de citocinas, dando início à resposta imune inata. Em modelos animais, o TLR2, TLR4 e TLR9 parecem estar relacionados com o reconhecimento de antígenos de Leishmania. A relação entre TLRs e leishmânia pode ser um mecanismo chave no desenvolvimento da doença ou no controle da mesma. Até o momento não existem estudos de TLRs na leishmaniose cutânea humana. Objetivo: Determinar o padrão de expressão e as células associadas com o TLR2, TLR4 e TLR9 na leishmaniose cutânea. O objetivo secundário é correlacionar a quantidade de TLRs com a quantidade de citocinas e células inflamatórias na pele. Métodos: Cem biópsias de pacientes com leishmaniose cutânea causadas por Leishmania (V.) braziliensis foram selecionadas inicialmente. Apenas os casos confirmados (presença de amastigotas no raspado, teste de Montenegro positivo, imunoistoquímica com presença de antígenos de Leishmania e reação em cadeia da polimerase com DNA de Leishmania (V.) braziliensis foram incluídos. Um grupo controle de pele normal foi incluído para comparação (quatro casos). A expressão de TLR2, TLR4 e TLR9 foi determinada por técnica imunoistoquímica, da mesma forma que os fenótipos celulares (células NK, macrófagos, células dendríticas, células CD4 e CD8) e citocinas (IL-1, IL-6, IL-12, TNF-alfa, IFN-gama). Dupla-marcação foi realizada para identificar as células que expressaram os TLRs analisados. Análise semi-quantitativa foi utilizada para avaliação da expressão de TLRs na epiderme, enquanto na derme foi realizada análise quantitativa. O nível de significância foi estabelecido com p<0,05. Resultados: Doze casos preencheram os critérios de inclusão. Os pacientes eram todos masculinos, com lesões apenas em membros inferiores e mediana de idade de 23 anos [16-47]. A expressão de TLR2, TLR4 e TLR9 na epiderme da pele normal foi alta. Quando comparados com pele normal, tanto TLR4 quanto TLR2 mostraram menor expressão no epitélio dos pacientes com leishmaniose e não houve expressão de TLR9. A média de células expressando TLR2 na derme foi de 136,36±82,46 células/mm2, ao passo que a média de células expressando TLR4 foi de 3,21±4,11 células/mm2. A contagem de TLR9 foi de 86,15±88,36 células/mm2 predominando em áreas de formação de granulomas. A regressão linear não demonstrou relação entre a contagem de células marcadas ou citocinas com TLR2 ou TLR4. O aumento proporcional da expressão de TLR9 relacionou-se com maior expressão de IL-12 e IL-4 (p < 0,05). A dupla marcação demonstrou que os macrófagos expressaram TLR2. A dupla marcação não mostrou expressão de TLR2 nas células dendríticas e nas células NK. Conclusão: A leishmaniose cutânea localizada associa-se com a presença de TLR2, TLR4 e TLR9. No epitélio a expressão de TLR2 e TLR4 em pacientes com leishmaniose está diminuída em relação aos pacientes controles. A expressão do TLR2 na derme é estatisticamente maior que a de TLR4 e TLR9, a qual é expressa pelos macrófagos. A expressão de TLR9 ocorre principalmente nas áreas de granulomas havendo relação com a expressão de IL-12 e IL-4
Introduction: One of the first systems of defense against microorganisms is the Toll-like receptors (TLRs) pathway. The activation of these receptors promotes the cytokine synthesis, initiating the innate immune response. In animal models, TLR2, TLR4 and TLR9 appear to be related to the recognition of antigens of Leishmania. The relationship between TLRs and Leishmania can be a key mechanism in the development of the disease or it control. Until now, there are not studies about TLRs in human cutaneous leishmaniasis. Objective: To determine the expression pattern and the cells associated with TLR2, TLR4 and TLR9 in cutaneous leishmaniasis. The secondary objective is to correlate the amount of TLRs with the amount of cytokines and inflammatory cells. Methods: One hundred biopsies from patients with cutaneous leishmaniasis caused by Leishmania (V.) braziliensis were initially selected. Only confirmed cases of cutaneous leishmaniasis were included in the analysis (presence of amastigotes in the scraping, positive Montenegro test, immunohistochemistry with the presence of Leishmania antigens and polymerase chain reaction with DNA from Leishmania (V.) braziliensis. A control group (4 cases) of normal skin was included for comparison. The expression of TLR2, TLR4 and TLR9 was determined by immunohistochemistry, as well as cell phenotypes (NK cells, macrophages, dendritic cells, CD4 and CD8) and cytokines (IL-1, IL-6, IL-12, TNF-alpha, IFN-gamma). Double-staining was used to determine the cells expressing TLRs. Semi-quantitative analysis was used for evaluation of the expression of TLRs in the epidermis. Quantitative analysis was performed to evaluate the expression in the dermis. The level of significance was defined as p <0.05. Results: 12 cases fulfilled inclusion criteria. The patients were all male, with lesions in lower limbs and median age of 23 years [16-47]. The expression of TLR2 and TLR4 in the epidermis of normal skin was high. When compared with normal skin, TLR2 and TLR4 showed lower expression in the epidermis. There was no expression of TLR9 in the epidermis in cases of cutaneous leishmaniasis and normal skin. The mean number of cells expressing TLR2 in the dermis was 136.36±82.46 cells/mm2, while the average of cells expressing TLR4 was 3.21±4.11 cells/mm2. The count of TLR9 was 86.15±88.36 cells/mm2, and it was found mainly in the areas of granuloma formation. Linear regression showed no relationship between the number of labeled cells or cytokines with TLR2 or TLR4. There was an association between TLR9 and two cytokines (IL-12 and IL-4). This correlation suggested that the proportional increase in the expression of TLR9 was related to greater expression of IL-12 and IL-4 (p<0.05). The double staining showed that macrophages and endothelial cells expressed TLR2. The double staining showed no expression of TLR2 in dendritic cells and NK cells. Conclusion: The localized cutaneous leishmaniasis associated with the presence of TLR2, TLR4 and TLR9. The expression of TLR2 in the dermis was statistically greater than that of TLR4 and TLR9, which is expressed by macrophages. The expression of TLR9 occurs primarily in the areas of granulomas was associated with the expression of IL-12 and IL-4

Orientadores: Maria Heloisa Souza Lima Blotta, Ronei Luciano Mamoni
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: A paracoccidioidomicose é uma micose sistêmica causada pelo fungo dimórfico Paracoccidioides brasiliensis, que pode apresentar-se sob diferentes formas clínicas, dependendo da resposta imunológica do hospedeiro. Trabalhos recentes têm demonstrado a importância da resposta inata no direcionamento da resposta adaptativa. Células do sistema imune inato reconhecem patógenos por meio de receptores de reconhecimento padrão (PRRs). Dentre esses, podemos destacar a família dos receptores do tipo Toll (TLRs) e dos receptores de lectina do tipo C (CLRs), que possuem membros capazes de reconhecer antígenos fúngicos. Nosso trabalho teve por objetivo avaliar a expressão de TLR-1, TLR-2, TLR-4 e dectina-1 (CLR) em macrófagos e neutrófilos de indivíduos saudáveis, após exposição a células leveduriformes das cepas de alta (Pb 18) e baixa (Pb265) virulência de P. brasiliensis. Como controle positivo foram utilizados os ligantes específicos para TLR-4 (LPS) e TLR-2 e dectina-1 (zimosan). A análise por citometria de fluxo revelou a redução da expressão de TLRs e dectina-1, mais evidente em monócitos do que em neutrófilos e após 30 minutos de estimulação, indicando que o reconhecimento dos antígenos fúngicos resulta na rápida internalização dos receptores. Houve uma tendência a um aumento da expressão relativa do RNAm de TLR-2 e dectina-1 em resposta à estimulação fúngica, em especial à cepa Pb265. A análise da produção de citocinas (RNAm e proteína) mostrou que as células fúngicas induzem a produção de citocinas inflamatórias e anti-inflamatórias, mas razão TNF-a/IL-10 diminuída em resposta ao zimosan e leveduras Pb265 indica uma produção relativa maior IL-10 nestas condições, enquanto que o estímulo com Pb18 privilegia a produção de TNF-a. Leveduras de P. brasiliensis também estimulam a elevada produção de PGE2 tanto por macrófagos como por neutrófilos, indicando a importância deste mediador na relação parasita-hospedeiro. Em conjunto, nossos resultados sugerem a participação do TLR-2, TLR-4 e dectina-1 no reconhecimento e internalização do fungo, e conseqüente ativação da resposta imunológica ao P. brasiliensis. Além disso, no caso da infecção por Pb265 a indução de uma resposta inflamatória exacerbada seria contrabalançada pela maior produção de IL-10, o que levaria a um melhor controle da infecção pelo hospedeiro.
Abstract: Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin America caused by the dimorphic fungus Paracoccidioides brasiliensis. The pattern of the immune responses to P. brasiliensis determines the disease progression and clinical outcome. Innate immune response is mediated by phagocytic cells, such as macrophage and neutrophils, which ingest and kill invading pathogens and then trigger the adaptive immune system through the secretion of cytokines and chemokines. The C-type like lectin receptors (CLR) and Toll-like receptors (TLRs) are the two main PRRs for fungal cells. The purpose of the present study was to evaluate the expression of TLR-1, TLR-2, TLR-4 and dectin-1 (CLR) in monocytes and neutrophils from healthy individuals after stimulation with Pb18 (high virulence) and Pb265 (low virulence) yeasts of P. brasiliensis. As positive controls we used specific ligands to TLR-4 (LPS), TLR-2 and dectin-1 (zymosan). Our results demonstrated a decreased of TLRs and dectin-1 expression more evident on monocytes than on neutrophils as soon as 30 minutes after yeast cells stimulation. This decrease was similar to the one caused by zymosan stimulation and indicates that up binding the complexes are rapidly internalized. There was a tendency towards an increased TLR2 and dectin-1 mRNA expression in response to fungal cells, mainly Pb265. P. brasiliensis yeast cells induced the production of proinflammatory and antinflammatory cytokines (mRNA and protein) but the low ratio between TNF-a and IL-10 in response to zymosan and Pb265 point out to a preferential production of IL-10, while Pb18 predominantly induced TNF-a secretion. P. brasiliensis yeast cells also induced an elevated PGE2 production by monocytes and neutrophils showing the important role of this mediator in fungushost relationship. Altogether our results suggest the participation of TLR2, TLR-4 and dectin-1 in P. brasiliensis recognition and internalization and consequent activation of the immune response against the fungus. Moreover, concerning Pb265 stimulation the induction of an exacerbate inflammatory response could be counterbalanced by an increased IL-10 production, resulting in an efficient control of the infection by the host.
Mestrado
Ciencias Biomedicas
Mestre em Ciências Médicas

Orientador: Dagmar Ruth Stach Machado
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A reação biológica as partículas de desgaste é crítica para indução da perda asséptica do implante mediante a osteólise. Deste modo, foi objetivo deste trabalho avaliar o envolvimento dos receptores Toll-like receptors 2 e 4 no reconhecimento das partículas de titânio e zircônia. Mensurados em cultura de macrófagos murinos desafiados com as partículas de zircônia ou de titânio comparando a expressão de TLRs, seus adaptadores intracelulares e citocinas pró-inflamatórias. Em in vivo foram estudados a indução da osteolise utilizando o modelo de calvária e a geração da resposta inflamatória através da indução do edema e hiperalgesia. As partículas são prontamente fagocitadas pelos macrófagos em cultura, e resultam no aumento da expressão de RNAm para TLRs 2, 3, 4 e 9, os seus adaptadores MyD88 e NF-kB e das citocinas TNF-?, IL-1? e IL-6. Contudo, o padrão de expressão de RNAm para TLRs entre as partículas é distinto, enquanto a zircônia induz um aumento significativamente na expressão de TLR2, o titânio modula a expressão significativamente maior de TLR3, TL4 e TLR9, respectivamente. Todavia, a expressão do RNAm para a molécula adaptadora MyD88 envolvida na sinalização intracelular de TLR é estimulada em ambas as partículas e com uma cinética de expressão semelhante. O fator de transcrição NF-kB necessário para efetuar a expressão gênica das citocinas envolvidas na resposta inflamatória apresenta uma cinética de expressão distinta entre as partículas, na zircônia a expressão é imediata e alcança o máximo da expressão após duas horas, enquanto as partículas de titânio induzem um aumento exponencial do fator de transcrição. A expressão de RNAm das citocinas inflamatórias TNF-?, IL-1? e IL-6 induzidas pelas partículas de zircônia é significativamente menor em comparação com as partículas de ix titânio. Contudo a expressão protéica da citocina TNF-? é maior nas em cultura de macrófagos expostas as partículas de zircônia, enquanto as partículas de titânio induzem a expressão protéica das citocinas inflamatória IL- 6. Ambas as partículas são capazes de induzir osteólise no modelo da calvária, contudo, a osteolise induzida pela zircônia assim como a perda óssea foi significativamente menor em comparação com as partículas de titânio. Assim embora ambas as partículas induzam edema e hiperalgesia nos animais de experimentação contudo as partículas de titânio uma maior sensação de hiperalgesia. Com base nos nossos resultados sugerimos que a biocompatibilidade da zircônia é maior em comparação com o titânio, e a perda asséptica é modulada pelo reconhecimento mediado pelos TLRs os quais ativam as vias de sinalização intracelular como os fatores de transcrição NF-kB levando a expressão de citocinas inflamatórias
Abstract: The biological reaction to wear debris is critical to the osteolysis underlying aseptic loosening of prosthetic implants Therefore was the aim of this study was to evaluate the involvement of Toll-like receptors 2 and 4 in the recognition of titanium and zirconia particles. Measured in cultured murine macrophages challenged with particles of zirconia or titanium by comparing the expression of TLRs, their intracellular adaptors and the proinflammatory cytokines. Particle-induced osteolysis was evaluated in mice calvaria model, whereas the inflammatory responses through induction of hind paw edema and hyperalgesia. The particles are readily phagocytized by macrophages in culture and result in increased expression of mRNA for TLRs 2, 3, 4 and 9, its adaptors MyD88 and NF-kB, TNF-?, IL- 1? and IL-6 . However, the pattern of mRNA expression TLRs is distinct for the particles, while the zirconia induces a significant increase in expression of TLR2; titanium modulates the expression significantly greater TLR3, TLR9 and TL4, respectively. However, the mRNA for the adaptor molecule MyD88 involved in intracellular signaling of TLR is stimulated in both particles and with a similar kinetic. The transcription factor NF-kB is needed to carry the gene expression of cytokines involved in the inflammatory response has a different kinetics expression between the particles, whereas zirconia induces the expression with the maximum after two hours of incubation, the titanium particles induce an exponential increase. The mRNA expression of the inflammatory cytokines TNF-?, IL- 1? and IL-6 induced by the particles of zirconia is significantly lower compared with the particles of titanium. However, the protein expression of TNF-? is greater in cultured xi macrophages exposed to the particles of zirconia, while the titanium particles to induce protein expression of inflammatory cytokine IL-6. Both the particles induce osteolysis in the calvaria model however; the induced osteolysis by zirconia as well as the bone loss was significantly lower compared with titanium particles. So although both particles induce edema and hyperalgesia in animal models, titanium particles induced greater sense of hyperalgesia. Based on our results, we suggest that the biocompatibility of the zirconia is greater in comparison with the titanium and the loss is modulated by aseptic recognition mediated by TLRs which activate the intracellular signaling pathways as transcription factors NF-kB and leads to expression of inflammatory cytokines
Doutorado
Imunologia
Doutor em Genetica e Biologia Molecular

Orientador: Wilson Nadruz Júnior
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: Estudos experimentais revelaram que a inibição de componentes da via de sinalização celular regulada pelos Toll-Like Receptors (TLRs) pode atenuar a hipertrofia cardíaca em animais submetidos à sobrecarga pressora. O objetivo deste estudo foi investigar a influência dos polimorfismos Asp299Gly do TLR4 e Pro249Ser do TLR6 sobre a estrutura do ventrículo esquerdo (VE) em indivíduos hipertensos. Foram estudados 443 pacientes por meio de avaliação clínica, laboratorial e ecocardiográfica, enquanto que os polimorfismos foram detectados por reação de polimerase em cadeia/enzima de restrição. Ademais, monócitos obtidos de sangue periférico de pacientes hipertensos foram estimulados in vitro com LPS (agonista de TLR4) e zimosan (agonista de TLR6) e a produção de interleucina-6 e fator de necrose tumoral-alfa (TNF-alfa) foi avaliada de acordo com a presença da variante Asp299Gly do TLR4 e Pro249Ser do TLR6. Mulheres que carregavam o alelo TLR4 299Gly apresentaram menor espessura de parede posterior do VE, menor espessura de septo, menor índice de massa do VE e reduzida prevalência de hipertrofia cardíaca. Mulheres homozigotas para TLR6 249Ser apresentaram menor espessura da parede do VE e menor espessura relativa da parede do VE em relação às mulheres com os genótipos Pro/Ser e Pro/Pro. Estes achados foram confirmados por análise de regressão linear múltipla, que incluiu idade, pressão arterial sistólica e diastólica, índice de massa corpórea, menopausa, diabetes mellitus e uso de antihipertensivos como fatores confundidores. Por fim, estudos funcionais in vitro revelaram que monócitos de homens e mulheres que apresentaram o alelo TLR4 299Gly tiveram menor produção de interleucina-6 após estímulo com LPS e a presença do alelo TLR6 249Ser em homozigose esteve associada a uma menor produção de interleucina-6 e TNF-alfa após estímulo com zimosan apenas nos monócitos extraídos de mulheres hipertensas. De maneira geral, esses dados sugerem que pode haver uma interação entre gêneros, polimorfismos dos receptores Toll-like e fenótipo do VE em indivíduos hipertensos. Sob esta perspectiva, estudos longitudinais são necessários para avaliar o impacto destes polimorfismos sobre o risco cardiovascular em mulheres hipertensas
Abstract: Experimental studies have shown that inhibition of components of cell signaling pathway regulated by Toll-Like Receptors (TLRs) can reduce cardiac hypertrophy in animals subjected to pressure overload. The aim of this study was to investigate the influence of the polymorphisms TLR4 Asp299Gly and TLR6 Pro249Ser on the structure of the left ventricle in hypertensive subjects. We studied 443 patients by clinical, laboratory and echocardiographic, while polymorphisms were detected by the polymerase chain / restriction enzyme. Moreover, monocytes obtained from peripheral blood of hypertensive patients were stimulated in vitro with LPS (TLR4 agonist) and zymosan (TLR6 agonist) and the production of interleukin-6 and tumor necrosis factor-alpha (TNF-alpha) was assessed according to the presence of the Asp299Gly variant of TLR4 and Pro249Ser of TLR6. Women who carried the TLR4 299Gly allele had lower posterior wall thickness, thinner septum, a lower rate of left ventricular mass and reduced prevalence of cardiac hypertrophy. Women homozygous for TLR6 249Ser had lower posterior wall thickness and relative wall thickness, compared to women with the genotype Pro/Ser and Pro/Pro. These findings were confirmed by analysis of multiple linear regression that included age, systolic and diastolic blood pressure, body mass index, menopause, diabetes mellitus and use of antihypertensive drugs as confounding factors. Finally, in vitro functional studies revealed that monocytes from men and women who harbored the TLR4 299Gly allele had lower production of interleukin-6 after stimulation with LPS and the presence of homozygous 249Ser allele was associated with a lower production of Interleukin-6 and TNF-alpha after stimulation with zymosan only in monocytes hypertensive women. These data suggest that there may be an interaction between gender, polymorphisms of Toll-like receptors and ventricular phenotype in hypertensive subjects. In this regard, longitudinal studies are needed to assess the impact of these polymorphisms on the cardiovascular risk of hypertensive women
Doutorado
Clinica Medica
Doutor em Clínica Médica

Estudos recentes têm demonstrado uma participação importante do receptor toll-like 4 (TLR4) na evolução de doenças envolvendo desordens metabólicas, como a doença do fígado gorduroso não-alcoólico (NAFLD). No entanto, as alterações do metabolismo lipídico que poderiam ser influenciadas pela ativação do TLR4 são desconhecidas. Neste estudo propomos caracterizar o papel do receptor TLR4 no metabolismo de lipídios no fígado de camundongos deficientes para o receptor de LDL, um modelo que desenvolve NAFLD quando submetido a uma dieta rica em gordura saturada e colesterol. Camundongos controle (C57 black6), deficientes para o receptor de LDL (LDLrKO), deficientes para o receptor TLR4 (TLR4KO) ou deficientes para ambos (duplo KO) receberam dieta controle ou hiperlipídica por quatro, oito ou doze semanas. Após o tratamento e sacrifício dos animais, avaliamos o perfil de lipídios plasmáticos, o conteúdo de lipídios do fígado e a expressão gênica de enzimas relacionadas à síntese e degradação de triglicerídeos (TG) e colesterol no fígado. O perfil inflamatório no fígado também foi avaliado. A dieta hiperlipídica induziu uma hipertrigliceridemia e hipercolesterolemia nos animais LDLr KO e duplo KO, sendo que o grupo duplo KO apresentou níveis séricos inferiores de triglicérides (TG) e ácidos graxos livres a partir de oito semanas de tratamento em comparação aos animais LDLrKO. A dieta hiperlipídica também induziu um aumento significativo no conteúdo de TG e de colesterol no fígado de todos os grupos. Na análise da expressão gênica não foram encontradas diferenças na expressão de proteínas relacionadas à síntese de triglicérides e colesterol (ApoB100, MTTP, GPAT1 e GPAT4) entre os grupos. Porém houve aumento significativo na expressão de proteínas relacionadas à oxidação de ácidos graxos (CPT1, MTP, ACOX, PBE, tiolase) e à síntese de ácidos biliares (CYP7a1) no grupo duplo KO em comparação ao grupo LDLr KO. No perfil inflamatório, a expressão de F4/80 demonstrou infiltração de macrófagos significativamente elevada no grupo LDLrKO tratado com a dieta hiperlipídica comparada a todos os outros grupos. No entanto, houve maior expressão de IL-6, IL-1beta e TNF-alfa no grupo duplo KO em comparação ao grupo LDLr KO. Nossos dados sugerem que a ativação do TLR4 no fígado de animais alimentados com uma dieta hiperlipídica pode contribuir para o acúmulo de lipídios e início da esteatose hepática. Estratégias para a inativação hepática do TLR4 podem diminuir a NAFLD não somente devido a diminuição da inflamação, mas por aumentar a oxidação de ácidos graxos no fígado
Recent studies have shown an important role of toll-like receptor 4 (TLR4) in the evolution of diseases involving metabolic disorders, such as non-alcoholic fatty liver disease (NAFLD). However, changes in lipid metabolism regulated by TLR4 activation are still unknown. In this study, we characterized the role of TLR4 receptor in hepatic lipid metabolism of mice deficient for the LDL receptor, a model that develops NAFLD when exposed to a diet rich in saturated fat and cholesterol. We investigated the role of TLR4 activation in the pathogenesis of diet-induced NAFLD by crossing LDLr KO mice with the TLR4 knockout mice (double KO). Animals were fed for 4, 8 or 12 weeks with high-fat diet (HFD) containing 18% saturated fat and 1.25% cholesterol. We evaluated plasma lipid profile, hepatic lipid content and gene expression of enzymes related to the synthesis and degradation of triglycerides and cholesterol in the liver. Liver inflammatory status was also investigated. We observed that HFD induced hypertriglyceri-demia and hypercholesterolemia in LDLr KO and double KO mice, but double KO animals presented lower serum levels of triglycerides and free fatty acids after eight weeks of treatment. HFD also induced a significant increase in liver contents of triglycerides (TG) and of cholesterol in all groups. We did not find differences in the expression of proteins related to triglycerides and cholesterol synthesis (ApoB100, MTTP, GPAT1, GPAT4) between the groups. However, we observed a significant increase in the expression of proteins related to fatty acid oxidation (CPT1, MTP, ACOX, PBE, tiolase ) and bile acid synthesis (CYP7a1) in double KO group in comparison to LDLr KO. Regarding the inflammatory process, F4/80 expression was elevated in LDLr KO mice fed HFD when compared to all groups. On the other hand, IL-6, IL-1beta e TNF-alfa expression was induced by HFD only in double KO mice. Taken together, our results show that TLR4 activation in liver from mice fed on a high-fat diet may contribute to lipid accumulation and steatosis onset. Strategies regarding localized TLR4 inactivation may increase the oxidation of fatty acids and improve NAFLD not only due to decreased inflammation

Os fibroblastos são atualmente considerados componentes ativos da resposta imune porque estas células expressam receptores do tipo Toll (TLRs), são capazes de reconhecer padrões moleculares associados a patógenos e mediar a produção de citocinas e quimiocinas durante a inflamação. A resposta imune inata do hospedeiro a lipopolissacarídeos (LPS) de Porphyromonas gingivalis é incomum, já que diferentes estudos relataram que este LPS pode ser um agonista para TLR2 e um antagonista ou agonista para TLR4. A sinalização por TLRs envolve proteínas adaptadoras, como MyD88 e TRAM, que são necessárias para a transdução do sinal até o núcleo para que ocorra a transcrição de RNAm para os mediadores da inflamação. O objetivo deste estudo foi investigar e comparar se a sinalização por meio de TLR2 ou TLR4 poderia afetar a produção de Interleucina (IL)-6, IL-8 e CXCL12 em fibroblastos humanos gengivais (HGF) e fibroblastos humanos de ligamento periodontal (HPLF). Objetivamos também comparar a participação das moléculas adaptadoras MyD88 e TRAM na expressão do RNAm dos mesmos alvos. Material e Métodos: Após silenciamento mediado por RNA de interferência de TLR2, TLR4, MyD88 ou TRAM, confirmado por RT-qPCR, HGF e HPLF, provenientes de três dadores voluntários, foram estimulados com LPS de Porphyromonas gingivalis ou com dois agonistas sintéticos de TLR2, Pam2CSK4 e Pam3CSK4, por 6 horas. A expressão do RNAm e das proteínas IL-6, IL-8, e CXCL12 foram avaliados por qRT-PCR e ELISA, respectivamente. Resultados: A expressão do RNAm de TLR2 foi regulada em HGF, mas não em HPLF por todos os estímulos. O silenciamento de TLR2 diminuiu IL-6 e IL-8 em resposta ao LPS de P. gingivalis, Pam2CSK4 e Pam3CSK4 de maneira semelhante, em ambas as subpopulações de fibroblastos (p<0,05). Por outro lado, a produção de CXCL12 permaneceu inalterada pelo silenciamento de TLR2 ou TLR4. No caso do silenciamento de MyD88 e TRAM, em ambos os subtipos de fibroblastos, o RNAm para os mesmos alvos também teve sua expressão diminuída (p<0,05). Já a expressão constitutiva de CXCL12 foi aumentada com o silenciamento de MyD88 ou TRAM (p<0,05). Conclusão: Estes resultados sugerem que a sinalização por meio de TLR2, por fibroblastos, as células residentes mais numerosas em gengiva e ligamento periodontal, pode controlar a produção de IL-6 e IL-8, que por sua vez contribuem para a patogênese periodontal, mas não interfere nos níveis de CXCL12, uma quimiocina importante no processo de reparação. Concluímos também que o silenciamento de MyD88 ou TRAM é capaz de diminuir o aumento da transcrição gênica de IL-6 e IL-8 provocado por LPS de P. gingivalis, embora a expressão constitutiva de CXCL12 seja regulada positivamente.
Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen associated molecular patterns and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual in that different studies have reported that it can be an agonist for TLR2 and an antagonist or agonist for TLR4. TLRs signaling pathway involves adaptor proteins, like MyD88 and TRAM, which are crucial for signal transduction to the nucleus and mRNA expression of inflammatory mediators. This study investigated and compared whether signaling through TLR2 or TLR4 could affect the production of IL-6, IL-8 and CXCL12 in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPLF). The role of MyD88 and TRAM on the mRNA expression of the same targets were also evaluated. Methods: After small interfering RNA-mediated silencing of TLR2, TLR4, MyD88 or TRAM, confirmed by RT-qPCR, HGF and HPLF from three volunteer donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12 mRNA expression and protein production were evaluated by RT-qPCR and ELISA, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, Pam2CSK4 and Pam3CSK4 in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. For MyD88 or TRAM silencing, IL-6 and IL-8 mRNA were also decreased, in both fibroblasts subtypes. However CXCL12 mRNA constitutive expression was increased by siMyD88 or siTRAM. Conclusion: These results suggest that signaling through TLR2 by fibroblasts, the most numerous resident cells in gingiva and periodontal ligament, may control the production of IL-6 and IL-8, which in turn contribute to periodontal pathogenesis, but does not interfere with CXCL12 levels, an important chemokine in the repair process. Also, MyD88 or TRAM knockdown may decrease the IL-6 and IL-8 LPS-induced upregulation and increase the constitutive CXCL12 mRNA.

La mucosa del tracte gastrointestinal (TGI) constitueix la superfície de contacte més àmplia de l’organisme amb el medi extern. A la llum del TGI, la barrera epitelial permet la separació entre els bilions de microorganismes residents i els immunòcits. En aquest context, el manteniment de la tolerància a la microbiota resident és essencial per a preservar l’homeòstasi; qualsevol esdeveniment que causi la desregulació d’aquestes interaccions pot desencadenar respostes pro-inflamatòries com les observades a la malaltia inflamatòria intestinal (MII). Els receptors Toll-like (TLR) regulen el diàleg entre l’hoste i la microbiota, i s’han associat a la patogènesi de la MII. Tot i que inicialment foren descrits en immunòcits, la seva caracterització ha evolucionat ràpidament en els últims anys, i actualment s’accepta que els seus rols depenen àmpliament de les cèl·lules que els expressen. Aquest treball tracta diferents aspectes de la funció dels TLRs en el TGI, oferint una visió integrada del seu paper en diversos tipus cel·lulars i condicions particulars. La nostra investigació s’ha centrat en com l’estimulació dels TLR2/4/9 influencia les respostes de diferents tipus cel·lulars del TGI inferior en condicions fisiològiques i d’inflamació. Amb aquesta finalitat, hem estudiat l’expressió i distribució dels TLRs en un model murí de colitis induïda per dextrasulfonat sòdic (DSS), així com els efectes de l’administració intracolònica de diferents dosis de lligands dels TLR2/4. També hem avaluat el paper potencial dels TLR2/4/9 en cultius de sistema nerviós entèric (SNE) i cèl·lules enteroglials (CEG) en termes de producció de citocines, quimiotaxi i sensibilització de la producció de citocines en macròfags. Els nostres resultats demostren que els TLR2/4 tenen una àmplia expressió en condicions fisiològiques al TGI, i es troben incrementats durant la inflamació, especialment en colonòcits i immunòcits. L’administració intracolònica dels seus lligands en condicions fisiològiques no alterà els paràmetres clàssicament avaluats per al seguiment de la colitis. Per contra, la instil·lació de lipopolisacàrid (LPS) durant la inflamació amb un protocol específic atenuà els símptomes de colitis i reduí l’expressió dels TLR2/4 desregulats. El mecanisme efector sembla basat en afavorir la preservació epitelial promovent la proliferació de colonòcits. De fet, s’observà un increment de l’índex de preservació epitelial associat a augments en l’alçada de les criptes i en l’expressió de marcadors de proliferació en colonòcits de ratolins tractats amb DSS+LPS. Per altra banda, els nostres experiments també demostren que les CEGs expressen TLR4 funcional que activa la via de senyalització NF-κB post-estimulació amb LPS, induint l’alliberament de citocines i quimiocines, i incrementant la quimiotaxi d’immunòcits. Als cultius de SNE s’observaren respostes similars, malgrat que la presència de macròfags residents fa difícil quantificar la contribució de cada tipus cel·lular. Aquests cultius també expressen TLR2/9 funcionals, però no s’observaren respostes als seus lligands si no s’afegeixen en combinació amb LPS. De fet, l’estimulació dels TLR4/9 donà lloc a respostes sinèrgiques en la secreció de molècules solubles que després sensibilitzaren les respostes de macròfags, disminuint la seva producció de citocines pro-inflamatòries. Els resultats presentats en aquesta memòria contribueixen a millorar la comprensió de les funcions dels TLRs en el TGI inferior durant l’homeòstasi i la inflamació. Com a conclusió, el paper dels TLRs varia en funció del tipus cel·lular estimulat i el seu ambient. Algunes de les respostes dirigides pels TLRs, com les observades en cèl·lules epitelials, poden ser utilitzades per a modular la inflamació, però d’altres, com les de les CEGs, han de ser evitades per a prevenir l’exacerbació d’aquests processos. En aquest aspecte, la selectivitat és clau, i podria ser aconseguida a través d’una dosificació i uns protocols d’administració acurats.
The mucosa of the gastrointestinal (GI) tract is the widest surface of the organism exposed to the external milieu. The epithelial barrier keeps trillions of microorganisms self contained within the GI lumen and separated from the immune cells. In this system, preservation of tolerance to resident microbiota is essential to maintain homeostasis; indeed, any event causing a dysregulation of these relationships might trigger pro-inflammatory responses such as those observed in inflammatory bowel diseases (IBDs). As the main receptors mediating the interplay between the host and the microbiota, Toll-like receptors (TLR) have been associated with the pathogenesis of IBD. Although initially described in immunocytes, knowledge regarding TLR expression and function has rapidly evolved in recent years, and it is currently accepted that their functions depend thoroughly on the cell type they are expressed in. The aim of this work was to approach some aspects of the function of TLRs in the GI tract, in an attempt to offer an integrated view on their role in different cell types in particular conditions. Specifically, our work has focused on how stimulation of TLR2/4/9 in different cell types populating the lower GI tract might influence their responses during homeostasis and inflammation. In order to achieve our objectives, we studied TLR expression and distribution in the context of the dextran sulphate sodium (DSS)-induced murine model of colitis, as well as the effects of intracolonic administration of different doses of TLR2/4 ligands. In addition, we assessed the putative roles of TLR2/4/9 in the enteric nervous system (ENS) and enteroglial cell (EGC) cultures in terms of cytokine release, chemoattraction and subsequent priming of TLR-induced cytokine expression in a macrophage-like cell line. Our results show that TLR2/4 display a wide expression thorough the lower GI tract in physiological conditions, and are up-regulated during inflammation, especially in colonocytes and immunocytes. Intracolonic administration of their ligands in physiological conditions had no apparent effects in the classical parameters used in assessment of colitis severity. Contrastingly, instillation of lipopolysaccharide (LPS) during inflammation in the described specific regime attenuated colitis severity and reduced expression of deregulated TLR2/4. The mechanism driving such effects seems to rely on increased epithelial preservation through induction of a proliferative response in epithelial cells, since higher epithelial preservation index was associated to increased crypt length and to enhanced expression of proliferation markers in colonocytes of DSS+LPS-treated animals. On the other hand, our findings additionally demonstrate that EGCs express functional TLR4 that activates the NF-κB signalling pathway after LPS challenge, inducing the release of cytokines and chemokines, and increasing chemoattraction of immunocytes. Similar responses were observed in ENS cultures, but the presence of resident macrophages in such cultures makes it difficult to quantify the participation of each cell type. ENS cultures had also functional TLR2/9, but no responses were observed to their ligands unless they were added in combination with LPS. Interestingly, upon TLR4/9 stimulation, synergistic responses were obtained in secretion of soluble molecules that subsequently primed the responses of macrophage-like cells, reducing their production of pro-inflammatory cytokines. The findings summarised in this manuscript contribute to improve the understanding of the functions that TLRs develop in the lower GI tract during homeostasis and inflammation. Overall, TLR roles may vary depending on the challenged cell type and its environmental situation. Some of the responses driven by TLRs can be used to modulate inflammation, such as those observed in epithelial cells, whereas some others must be avoided to prevent exacerbation of these processes (those in EGCs, for instance). Selectivity is the key, and might be achieved through accurate dosage and precise administration regimes.

Stimulation of Toll-like receptors (TLRs) in cells of the innate immune system activates the expression of a proinflammatory and antimicrobial gene program controlled by a network of transcriptional regulators. We show that NFAT5, which belongs to the Rel family of transcription factors and was previously characterized as an osmostress responsive factor, is required for the expression of a group of TLR-responsive genes in macrophages, such as Nos2, Il6 and Tnf. NFAT5 recruitment to its target genes is dependent on IKKβ activity, de novo protein synthesis and is sensitive to histone deacetylases. Interestingly, NFAT5 is essential in the response to low doses of TLR ligands, regulating specific gene subsets depending on the stimulus strength. We also show that macrophages use NFAT5 to facilitate chromatin accessibility, allowing the recruitment of transcriptional regulators such as p65/NF-κB, c-Fos and p300 to its target regions. We use Nos2 as a gene whose induction is NFAT5-dependent especially at low doses of LPS to demonstrate that NFAT5 controls the recruitment of p65 by facilitating the activity of H3K27 demethylases, without influencing the binding of Polycomb repressive complex 2 or JMJD3. Altogether, this thesis characterizes NFAT5 as a novel regulator of the immune response to low pathogen load involved in the control of local chromatin accessibility.
En las células del sistema inmunitario innato, la estimulación de los receptores de tipo Toll (TLR) activa la expresión de un programa génico pro-inflamatorio y antimicrobiano que está controlado por una red de reguladores transcripcionales. Hemos demostrado que el NFAT5, perteneciente a la familia de factores de transcripción Rel y previamente caracterizado como un factor de respuesta a estrés osmótico, es importante para la expresión de un grupo de genes de respuesta a TLRs, entre ellos Nos2, Il6 y Tnf. El reclutamiento del NFAT5 a sus genes diana requiere la actividad de IKKβ, la síntesis de novo de proteínas y es sensible a la acción de las deacetilasas de histonas. Resulta interesante el hecho de que el NFAT5 es esencial para responder a bajas dosis de ligando de los TLRs, y que regula grupos de genes específicos dependiendo de la intensidad del estímulo. También mostramos que NFAT5 facilita la accesibilidad de la cromatina en macrófagos, permitiendo el reclutamiento de reguladores transcripcionales como p65/NF-kB, c-Fos y p300 a sus regiones diana. Utilizando Nos2 como un gen cuya inducción es más dependiente de NFAT5 a bajas dosis de LPS, demostramos que el NFAT5 controla el reclutamiento de p65 gracias a que facilita la actividad de las demetilasas de H3K27, pero sin influir en la unión del complejo Polycomb 2 ni JMJD3. En conclusión, esta tesis caracteriza al NFAT5 como un nuevo regulador del sistema inmunitario implicado en el control de la accesibilidad local de la cromatina en respuesta a baja carga de patógenos.

Toll-like receptors (TLRs) are a group of trans-membrane proteins playing important roles as pattern-recognition receptors (PRRs) of innate immunity that are mainly expressed on professional antigen presenting cells (APCs). Stimulation of APC with TLR ligands, a variety of Jnolecular components derived from microorganisms, triggers innate immunity and establishes adaptive immune responses. I studied the impact of TLR specific for bacterial components on antigen processing and presentation of protective antigen (PA) of B. anthracis by bone marrow macrophages from mice.

L’exposition aux rayonnements ionisants de la sphère abdomino-pelvienne est associée à une haute incidence de complications. Lors des cas les plus importants, l’apparition d’ulcérations peut entrainer le décès des patients en absence de lourds traitements. Dans ces cas, des essais cliniques sont réalisés avec des Cellules Souches Mésenchymateuses (CSM). Les radiothérapies abdomino-pelvienne peuvent provoquer à court et/ou long terme des effets délétères. Des études démontrent que l’injection de motifs bactériens confère une radioprotection au niveau intestinal. Ils stimulent des récepteurs (Toll-Like-Receptors (TLR)) situés à la surface des cellules intestinales. Cette thèse a pour but de caractériser les effets sur l’immunité et sur la réparation tissulaire de la stimulation des TLR dans un modèle d’irradiation colorectale localisée à 20 Gy (effets aigues des radiothérapies) chez le rat. Puis de potentialiser les effets des CSM avec une adjonction de ligands de TLR lors d’une irradiation colorectale localisée à 27 Gy (complications accidentelles). Ce travail a permis à 20 Gy, de montrer que la stimulation des TLR conduisait à améliorer l’homéostasie (normalisation des lymphocytes T, induction de lymphocytes T régulateurs (Treg) et de macrophages « anti-inflammatoire » M2). Dans le modèle 27 Gy, l’injection de ligand de TLR pré greffe de CSM permet une amélioration du climat immunitaire avec une diminution des cytokines pro-inflammatoires et l’induction de Treg et M2. Ces modulations pourraient permettre une meilleure implantation et efficacité des CSM. Toutes les observations apportées montrent que la stimulation de l’immunité est une approche pour limiter les dommages radio- induits
Exposure of the abdomino-pelvic sphere to ionizing radiation is associated with a high incidence of complications. Radiation therapy may cause short and / or long-term harmful effects. In the most severe cases and in the absence of heavy treatments, the appearance of ulcers may induce the death of patients. Clinical trials are being conduced with Mesenchymal Stem Cells (MSC) to cure theses complications. Others studies indicate that the injection of bacterial motifs limits the radiotoxicity in the intestine. They stimulate receptors (Toll-Like- Receptors (TLR)) located on the surface of epithelial and intestinal immune cells. The first aim of this doctoral work is to characterize the effects of TLR stimulation on immunity and tissue repair using a model of localized colorectal irradiation at 20 Gy (acute effects of radiotherapy) on a rat. The thesis then aims to potentiate the effects of the MSC treatment when adding TLR ligands upon localized colorectal irradiation at 27 Gy (accidental complications). This work, using a 20 Gy exposure, show that TLR stimulation improves homeostasis (normalization of T cells, induction of regulatory T cells (Treg) and macrophages "anti-inflammatory" M2). On the 27 Gy colorectal model, the injection of TLR ligand before CSM transplant improves the immune climate by reducing pro-inflammatory cytokines and inducting Treg and M2 cells. These modulations could contribute to improving the implantation and effectiveness of CSM. The observations have all shown that the stimulation of immunity is an approach to minimize radiation-induced lesions